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RNA Extraction - Science method

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So I'm using TRIzol for RNA extraction, but suddenly I'm getting no pellet during the isopropanol step *even though I added glycogen.*
A few weeks ago I:
1. Took a large quantity of bacteria
2. Resuspended in TRIzol
3. Made a bunch of aliquots
4. Stored them all at -80
And those couple weeks ago, I was getting~50 ug of RNA per aliquot (with good RINs, and they looked great on RNA gels).
Yesterday, I took another aliquot and tried to prep it using the exact same process, and got nothing. No pellet appeared during the isopropanol precipitation step even though I added GlycoBlue.
I continued the purification to see if the pellet was just hard to see: ~0 ng/uL by nanodrop
Did I just make a pipetting error? Was my isopropanol bad? No: I repeated the repeated the process *again* with completely new sample, completely new isopropanol, and still no pellet at all.
I'm confident I'm lysing my samples well. I optimized the lysis a while ago, but even before optimization, my yields were never this bad.
I'm sure I added the GlycoBlue. I watched it diffuse into my sample.
I'm sure I added isopropanol. I watched the alcohol/water mix and used brand new isopropanol.
I'm sure I mixed the isopropanol/aqueous phase. I watched it carefully.
I'm sure I actually spun my samples. I tried it over and over.
Protocol:
1. Take bacteria+TRIzol aliquot from -80 (which used to give ~50 ug)
2. Lyse via bead beating (same beads, same bead beater, same settings, same duration that gave 50 ug in the past)
3. Add 0.2 V chloroform
4. Centrifuge 12k xg for 15 min
5. Take upper, colorless aqueous phase
6. Add 1 V of RT isopropanol
7. Add ~30 ug GlycoBlue
8. Invert a bunch of times to mix well
9. Incubate 10 min RT
10. Centrifuge ~20k xg for 10 min
Nothing. No pellet at all. Even with glycogen.
I tried spinning again: No pellet
How is this possible?
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Hi Gen,
Doing vortex after adding isopropanol to chloroform is necessary to get a pellet. Chloroform should be mixed well with isopropanol. But once you get a pellet, for washing steps be very careful, ethanol should be added very gently to the tube. No vortex in this step.
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I would like to extract total RNA (including microRNAs) from frozen human skin biopsies embedded in Tissue-Tek (OCT Compound), but the literature is pretty scarce when it comes to this combination. The only paper that can lend me any hand is attached to this topic. Does anybody here have any experience with this?
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Try to read this article it may help.
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I am preparing CTAB buffer and I read that addition of PVP (Polyvinylpyrrolidone) helps to remove phenolic compounds. Most of the times in protocols there is no additional information about average molecular weight of used PVP, but I found in some, where it was specified that it was PVP 40 (mol wt 40 000) for DNA extraction. But I wanted to know if I can use the same for RNA extraction or should I use PVP 360 (mol wt 360 000)?
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PVP is basically a polymer, which will only mention as %. So its better to go with dilution. In my experience, CTAB-buffer with 2% PVP works well for the Isolation of DNA. Incubate your CTAB with PVP for 15 mins at 65 C and then vertex.
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Hi there,
I'm looking to compare the expression of a particular gene in fish brains across three developmental stages. My aim (as first step) is to use the entire brain for RNA isolation steps. This is simple with larval and juvenile stages, however, the RNeasy Lipid Kit has a maximum tissue weight of 100mg. Thus, it is far less easy to extract RNA representative of the entire brain in adult fish (with brains that are coin-sized).
So far, I've tried grinding the adult brain in liquid nitrogen using a large mortar and pestle, mixing the cold 'powder', then weighing and using 100mg of it in the kit - hoping that this sample comprises bits of tissue from all parts of the brain.
I worry, of course, that I'm not truly getting a true representation of the entire brain when I do this. Does anyone have any suggestions on what to do differently to get the same result?
Thank you in advance!
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What you are already doing sounds like a reasonable way to get even representation. To make it even better, you could prepare a liquid homogenate or lysate from the entire brain and then use a subsample of this liquid for your RNA extraction.
We routinely homogenise mouse brain tissue in PBS so that we have a liquid that evenly represents our brain region of interest that can be divided up for multiple experiments. We use glass dounce homogenisers and 500-1000uL of PBS for 15-50mg of tissue. I think you could probably 'fit' quite a bit more tissue into the same volume of PBS. When RNA quality is really really critical, we supplement the PBS with DTT (1uL/mL of 1M DTT) and RNaseOUT or Superasin (1uL/mL).
To maintain RNA quality, it is critical to keep your sample cold. We dissect on ice and store tissue samples at -80C. Tissue is thawed immediately before it is homogenised (it comes out of dry ice, into my hand just long enough to unstick the sample from the tube, and then directly into the homogeniser, one sample at a time) and solutions and equipment for homogenisation are kept ice-cold. Under these conditions we routinely extract RNA of RIN9+ from mouse brain.
Homogenate in PBS should be processed as a liquid sample (follow kit instructions for liquid sample when determining volume of lysis buffer etc.) Alternatively, after aliquoting the required amount of homogenate, you can centrifuge it at moderate speed (5000 * g) for 3-5 minutes at 4C and process the pellet as if it was tissue. I prefer not to do this for brain tissue because homogenising shears neuronal processes and these can end up in the supernatant, so you throw out some of the axonal and synaptic RNA.
Another option is to homogenise your tissue directly in lysis buffer, using the appropriate volume of buffer for the weight of tissue. Then, take an aliquot of lysate that suits your kit capacity, and process that. This is slightly better for your RNA quality, but will waste a lot of lysis reagent, which is why we don't do it (since the RNA quality with the PBS method is so high anyway.)
Hope this helps :)
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Why I lose my lettuce hairy roots RNA about 5 or 6 hours after extraction with licl method and on electrophoresis only it's DNA band will remain with no RNA?
I do the extraction and I get the RNA bands with a tiny small DNA with that; but after I used DNase and for one of my samples and I run it on gel with my RNA extraction sample wihout DNase;I saw no bands in the sample with Dnase and no RNA bands in the sample without DNase that I had the bands just a few hours ago when I run it on the gwl after extraction for the first time. I don't know why I lost my RNA? I keep all the things on ice too,and keep the sample in -20 at the first hours of extraction,but then keeping it on -40.
and I also want to know what will happen if I use DNase more? Does it effect my RNA Bands?!
and another more question... I did gene tranformation; and I get transgenic hairy roots of lettuce. Can I just do the RTpcr and cDNA synthesis without using DNase?!
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Thanks all of you for your help. :) They were great tips. ;) Thank you.
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Hi everybody,
Im trying to isolate RNA from umbilical cord total tissue by nitrogen freezing and powdering and then Qiazol/Choloroform extraction. I have obtained good results with placental total tissue but with cord the problem is that the pellet is very viscous, and when I try to solubilize it in water to quantify, I cannot even to pippete because of this.
Could anyone show me a protocol to do this?
Thanks
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Hi, Francisco,
did you solve the problem? I am having the same trouble, exactly like what you described, do you have a good method now?
thanks!
Ninuo
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Monarch® RNA Priming Buffer - any substitutes or Homemade Recipes? Qiagen Buffer RW1? We can't purchase this RNA Priming buffer because of the customs restrictions, so I am looking for any suggestions. Thanks a lot!
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Great, thanks a lot, Mira!
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I want to do transcriptomic study on 5dpf zebrafish larvae. The larvae will be exposed to toxic to see the differential gene expression between control and exposed zebrafish. What is the best way(s) to euthanize this larvae. Im afraid if using tricaine will affect the transcriptome and if using cold water, the larvae will experience hypoxic condition..please help
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MS-222 is an excellent method for larvae and it will not affect expression levels if you choose the proper dose and time
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Hi everyone,
According to your experiences, what are the best RNA extraction kits for FFPE tissue samples in terms of RNA quantity, quality, and process time? The downstream application will be RNA sequencing.
Thank you.
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If your lab has money, the Promega CSC/RSC kit for RNA FFPE extraction is the best. We have evaluated many column, bead, technologies and many vendors, and the Promega routinely outperforms as far as sample quality, quantity, and purity. If no money, any home-brew deparaffinization methods (xylene, mineral oil) will work, coupled with your standard Trizol extraction will yield superb quality and quantity of RNA. Best of luck!
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Im using a Nucleic Acid Detection Kit for Multiplex Real Time RT-PCR. Its content is:
- PCR Reaction Mix (Transcript II Multi probe one-step qRT-PCR SuperMix 1 UDG)
- PCR Reverse Transcriptase (RT plus RNase inhibitor)
- PCR Primer/Probe Mix (Primers and probes for ORF1ab and N genes; primers and probes for the control-RNase P gene)
- Positive Control (In vitro transcribed RNA with ORF1ab and N gene sequences; In vitro transcribed RNA with the control-RP gene sequence)
- Negative Control (Water)
Regarding I do not own a qPCR termocycler. Im performing a regular PCR and then run a electrohpresis gel. The product length for RP primers is 65pb and the product length for ORF1ab's primers is 129pb.
The protocol specified by the manufacturer is a one-step RT-PCR program:
1 cycle at 50°C for 5 min (Reverse Transcription)
1 cycle at 95°C for 30s (Pre-denaturation)
45 cycles of:
95°C for 5s (Denaturation)
60°C for 30s (PCR cycling)
The problem is that my positive control is not showing a positive result (I do not see a band in 129pb)
But the PCR works fine because I see bands in 65pb (RP gen) in the Samples wells.
I do not perform a DNA clean up in the extraction process. Should I do it? (Im using a ARN/ADN extraction kit with spin colunms, proteinase K, etc). So my sample has DNA and ARN.
My question is: Is it possible that the bands I see in 65pb in samples are produced by the primers bining to the original ADN (from the extraction) and not produced by primers binding to the cDNA? (which should be product of the Reverse Transcriptase transcribing the mARN to cDNA, and the syntetic ARN to cDNA too)
My assuption is that there is no cDNA then there is no bands in 126pb. Why there is no cDNA? Posible causes:
1) Reverse transcriptase is not working. Why? how to verify?
2) Positive Control ARN is degraded. Why? how verify?
3) Other issue? any ideas?
Thank you in advance!
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Hi, Donato.
You're more than welcome and I'm more than happy to know I could give some help.
Last but not least, If our support helped you, let us know if you got good results and share your final protocol. If it is not a problem. Ok?
All the best.
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In my previous lab, I routinely performed RNA extraction from yeast cells directly in hot phenol supplemented with SDS and acid-washed beads. This was enough to disrupt yeast cells which have a cell wall. Then I would do a round of chloroform extraction followed by precipitation with ammonium acetate and isopropanol. Exceptionally I would grind frozen yeast cell pellets in a mortar with liquid nitrogen, and use 8M guanodine isothiocyanate to disrupt proteins before extracting RNA.
I do not see why the same procedures would not work for mammalian cells; given that they do not even have a cell wall I suspect that beads would not even be necessary for the hot phenol procedure described above. However, most people seem to use TRizol (invitrogen) or Isogen (Wako Nippon Gene), which contain phenol and guanodine isothiocyanate in undisclosed proportions.
Is there a reason other than ease of use for why people prefer the TRizol procedure even though it is rather expensive? Would any of the two classic methods described above work just as well for mammalian cells?
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Josephine Galipon, it may not change your preferred RNA extraction methods, but the proportions of phenol and guanidine isothiocyanate in Trizol can be worked out from its patent (attached).
TBH, I think phenol can be as efficient as Trizol for RNA preparation. And Trizol doesn't give an RNA prep free of DNA contaminants either. I always use enzymatic digestion to rid of the contaminants in RNA and DNA preps, if needed.
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I would like to get rid of heparin in RNA extracted from heparinized plasma. Now I'm using heparinase II (Sigma H6512-10UN) dissolved in 100 uL of 100 mM sodium acetate pH 7.0.  I used 1, 2 or 3 uL of heparinase with approximately 1 ug of RNA and incubated at 37 C for 1 or 2 hours continuing with 1 step RT-PCR but didn't success.  Is there other protocol to get rid of heparin in RNA extracted from heparinized plasma by using heparinase?
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Hi Sirinart,
I would like you to share the protocol you eventually used to resolved this challenge. Thanks
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Hello everyone,
I want to measure the expression change of some mRNA, miRNA, and LncRNA at once under a specific condition.
Since I am going to do relative quantification, is there any RNA extraction reagent that can extract all RNA molecules simultaneously?
I am willing to hear if you have other solutions rather than a reagent for all molecules.
Thanks for your help.
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A cost effective method is extracting the total RNA from your samples all at once using biozol or triozol reagent, then using the corresponding primers of mRNAs, miRNAs and lncRNAs for qRT-PCR.
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When performing Viral RNA extractions I perform a negative extraction using water. This is then run as a negative control for my RT-PCR reaction. Recently this control has been showing up RNAse P positive. In addition, when I run the same extract negative sample in duplicate on my PCR plate, sometimes I get RNase P in only one of the duplicates. What is happening? It only ever happens in the Extraction Negative, it never occurs in my PCR negative control.
This issue is occurring for multiple technicians.
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Probably due to some contamination
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How biological materials (Animal carcasses, excrements) collected in field can be best stored for a later DNA and RNA extraction? what is the protocol to follwo and what are the storing equipements needed.?
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Hi Imad,
We use a "homemade" buffer in our expeditions, where freezing at -80 is not an option. It is called NAP buffer and preserves well DNA and ARN. Here is a link to the article: https://onlinelibrary.wiley.com/doi/pdf/10.1111/1755-0998.12108?casa_token=qgIKvJ4dV1kAAAAA:sEJG807qo-hjPXIc9J54AKwwkxiiqsZSWmFdTd91VobTRfivCVxUYECwRzyUByrGGabSVsPZHrdl. We usually collect tissue samples of liver from carcasses, when it is not fresh, muscle/heart works better, given that liver degrades quick. For mammal specimens that we collect, we skin them in situ and fix the skin in formalin for a few hours and then store it in ethanol 70%, but the skinned body goes directly to ethanol 70% so that there is still a good stock of DNA. For feces Carlos Dominguez Sarabia might know better.
Cheers,
Arlo
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Hi all! :)
I want to extract RNA from zebrafish embryos, 3dpf. My limitation will be the number of fish needed to gain enough material after (1) extracting total RNA (2) doing poly A selection. 
So I looked into RNeasy Micro Kit (Qiagen), miRNeasy Mini Kit (Qiagen) and Trizol (Ambio). The product has to be very high quality as it will be used for sequencing. 
Any input or experiences of
- How much material do one gain from e.g. 10 embryos day 3 with the different protocols?
- Which protocol is better in terms of quality/easy to use? It would be a great if I also got the DNA from my pooled sample so for that I guess Trizol but do anyone have any experiences with using that for zebrafish embryos? 
Suggestions for poly A selection is also appreciated. 
Best regards,
Sanna Gudmundsson
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Hi! Sorry that I am just getting back to you now. So after adding RLT buffer (+ betamercaptoethanol) I used a hand-held polytron homogenizer to completely disrupt the embryos before continuing with the rest of the RNA isolation. In my experience, the homogenization step is one of the most crucial to obtain a good RNA yield.
Hope this helps.
Andrew
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I'm looking for an alternative method from the Qiagen RNeasy Plant Mini kit to isolate total RNA from Cotton root tissue. 
I'm considering using the TRIZOL reagent to hopefully get a higher RNA yield, but I've read that TRIZOL extraction produces RNA with a high level of carbohydrates (and thus a low 260/230 ratio). This would produce a sample that is far too impure to perform cDNA synthesis, so it's important that whatever method I use either produces RNA isolates with a high 260/280 and 260/230 ratio after extraction, or yields a high enough RNA concentration that I can do multiple ethanol washes without loosing too much of my isolates.
Does anyone have any experience using the TRIZOL reagent to extract RNA from Cotton tissues, namely the root systems?
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Hi Walker Maffit , did you get a chance to make this work? I know your post has been a while. I am curious about what worked for you. I am researching RNA-extraction methods from cotton roots currently, would like to hear people's successful procedures.
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Hello
I am working on a project where I need to extract RNA from my samples. For some reason, Nanodrop gives me values below [10 ng/uL] for almost all my samples, even though I saw a pellet in all of them, so expected to get at least [100ng/uL]. Does anyone know why the values could be so low?
Another question relates to protein extraction from the same samples. After I had added chloroform and extracted the RNA from my samples, I was aiming to extract protein as well, but the protein layer seemed to be almost non-differentiated from the DNA layer in the sample, so I didn't know what to do with it. I simply froze the samples in -80, because it was getting very late. Does anyone know whether I would be able to extract the protein if I thaw my samples and attempt again or are the samples now pretty much destroyed because of freezing them with residual chloroform and without any handling before?
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Dear colleague:
I think you have problems with your RNA extraction. In my experience, when NanoDrop measurements are below 10 ng/uL, that measurements correspond to residual contamination. The pellet that you observed are just precipitated salts. Maybe you tissue sample is not enough, or your homogenization method is not the optimal for your tissue.
Regarding to your second question, we have performed the following protocol:
We have obtained very good results, but you must follow the protocol very closely.
I wish you the best!!!
Regards
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Hi All
I am using Trizol LS  reagent for RNA and DNA extraction from Grape-vine leaf tissue, but I couldn't find any RNA and DNA. I found big pellet and a bit it is hard to dissolve. I check the quality using nono drop and it seem there are some RNA but poor quality. When I check it using bio-analyzer, there is no RNA at all. Can any one help please? 
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Some simple tips from when I first started RNA isolation:
1. Make sure homogenized tissue is immediately placed in TRIzol buffer. If you are grinding with liquid nitrogen, be sure to not let the tissue thaw before being put in buffer. Even slight thawing will yield degraded RNA
2. Do not vortex. Ever. Invert instead (gently).
3. After the addition of isopropanol, invert 3-5 times. <seems simple but since I started inverting here I have had a 100% success rate with pellets.
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I am going to work with dithiothreitol (DTT) whilst isolating DNA and RNA, so I was looking for a safe alternative to a fume hood, such as maybe a specific type of mask or anything different that you might know of. Any help would be greatly appreciated. Thank you.
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I'm not sure what problems are presented by a fume hood. You can have walk-in ones as well as benchtop versions. If it is handling, then possibly a glove-box would be acceptable. In general, if something requires PPE for any reason, it is NOT a good idea to release it into any common areas.
Best regards,
Steven
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I am trying to extract RNA using trizol from very small parts of honeybees e.g. antennae and mouth-parts and was taking as much of the aqueous phase as possible in order to maximise yield. Following multiple problems with DNase treatments not working we decided that phenol carry over was to blame and are now trying to find a middle ground between decent quality (260/280 ~1.8-2 and 260/230 ~ 1.8-2) and decent yield. I have started using glycoblue as a co-precipitant to increase the yield and whilst the 260/280 remains high the 260/230 never gets above 1.3, is this the glycoblue?
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YES. It does affect, both concentration and 260/230 ratios. Change to a glycogen without any dye and you´ll see how your ratios go up by a lot.
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I use Glycoblue to dye my RNA pellets deriving from cytosolic or nuclear RNA (concentrations ranging from 150 to 600 ng/uL) and everything is pretty fine with the absorbance ratios from Nanodrop.
However, when I do the same on RNA extracted from heavy polysomes (concentrations ranging from 65 to 170 ng/uL) I get very low 260/230 ratios (<1).
Has anyone ever faced a similar problem?
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YES. It definitively does. I have been using glycoblue for a couple of months and obtaining really bad 260/230 ratios. I decided to use a glycogen without blue dye and the ratios went crazy up
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It looks almost as if the interphase is floating up into the aqueous phase during my extraction procedure. It doesn't form a distinct layer; it is very irregularly shaped and juts out into the aqueous phase at certain points so when I extract the RNA by removing the supernatant, I feel as if I am also getting part of the interphase. Sometimes it looks like the upper aqueous layer is mostly cloudy.
Has anyone else had any similar experiences? When I do the extraction with tissue, there is always a pretty clear/opaque looking interphase.
Whenever I measure my samples (diluted in water), my 260/280 ratio is always >2.5, so I feel as if there is DNA contamination since DNA is supposed to reside in the interphase.
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Did you end up figuring out what was the problem? I'm having a similar issue.
Thanks!
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Dear Researcher
I am working on passion fruit peel at ripening stages, and want to do qPCR but facing problem in getting good quality RNA from fruit peel,
I have tried to extract the RNA from the ripened fruit by using different RNA extraction kit from different companies, such as Qiagen, Takara, Bioflux, Genestar, and but the concentration is about 100-200 ul/ml, with very bad quality is such as A260/A230 = 1.3-1.60,
but by Trizol method, the concentration was about 600-800 ul/ml, but the quality was the same such as 1.3-1.70,
As I picked the sample from field myself 1 -1.5 months ago and transferred to Lab by keeping in 10 C, thermobox, and then peeled, dried into liquid nitrogen and kept -80 C,
while extraction, i have tried by taking the (powder) sample from 50 -200mg but all have same results,
looking for your suggestions,
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@hafiz Muhammad Rizwan,
I suggest why don't you a give a try to the Spin Column Method of RNA Purification? It's quite effecient in this regard.
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Hi, everyone!
I'd like to have some recommendations about soil RNA extraction.
I've tried some kits but I'm getting low concentration in the Qubit evaluation.
Does anybody have a kit to recommend or any idea about what could be helpful?
Thank you!
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We use this and get quiet good yields. We have just replaced the bead beating step and use TriZol for this step.
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I'm in the process of optimizing a protocol for the ethanol precipitation step of isolating RNA from fatty tissue. Could anyone recommend changes to my protocol to potentially improve the loss of RIN (RNA integrity as calculated by a Bioanalyzer) and drop in 260/280 that I see (decrease in RIN is usually 1.0 - 2.0, decrease in 260/280 of 0.1 - 0.2). Please advise!
1) Add ice-cold water to RNA sample
2) Add cold 3M Sodium acetate buffer to each sample
3) Add cold glycogen and mix
4) Add 2.5 parts ice-cold 100% EtOH (incubate 60'+, then centrifuge 10' at 16,000 x g and 4C, decant RNA)
5) Add 4.6 parts ice-cold 70% EtOH (vortex gently, centrifuge 5' at 7,500 x g)
6) Decant EtOH and air dry
7) Resuspend RNA in 15uL sterile nuclease-free water (store in -80*C)
8) re-assess RNA quality, 260/280, and RIN values
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After addition of sodium acetate and glycogen as per your protocol, add equal amount of isopropanol to your sample and mix by inverting the the up and down. Incubate on ice for 10min and then centrifuge at 14000 rpm at 4 degree for 15 min. RNA pellet will be obtained and discard the supernatant. Wash the pellet with 7% EtOH. air dry the pellet and then dissolve in DEPC water.
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Dear colleagues,
I have a bucket of carrot's inflorescence buds that were fixed with FAA, dehydrated and stored in methanol at -20°C for ca. 3 months. I did ISH with some of them that worked fine (so I assume RNA is there) but I decided to synthesize additional probes and I am running out of RNA stock. Could I rehydrate the samples, grind them in LN2 and proceed with Trizol isolation? Do you have any experience on that field?
I found some paper in which authors managed to extract good quality RNA from aquatic insects even from ethanol or acetone after prolonged to storage but plants are not exactly the same I suppose.
Best wishes
Jakub
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Hi, Jakub Baczyński , I met a similar challenge. I just fixed my plant materials (maize kernels) in FAA without doing any further processing, and I want to extract RNA from them... I was wondering if you have tried
David Farringdon Spencer
's method and how that works?
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During the course of the cultivation of a Pseudomonas sp. strain on a toluene derivative we observe a strong darkening of the media, supposedly because of polyphenolic (polycatecholic) compounds formed. The same obervation can me made sometimes while bacteria grow on aromatic compounds . 
We like to isolate RNA from the culture but obviously the cells and later the RNA strongly sticks to the polyphenolic/dark precipitation in the media. Does anyone has experience with that and a solution for that? (The RNA extraction from cells grown on succinate work perfectly.)
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Since I am working with carnivorous plants, which produce a high amount of polyphenols under stress, we had some problems with DNA extractions and PCR.
After some research, I found out some solutions, which may also work for you.
Since you are observing a strong darkening of the media, we can assume that you have oxidized compounds, probably caused by oxidases. If that is the case, you will need to change your extraction method from the beginning.
Some oxidases can rapidly cross-link compounds to RNA or in my case DNA, during the disruption of the cells, so you need to prevent this to happen.
The best way is to use liquid nitrogen before you disrupt the cells, this will inhibit the work of possible oxidases.
Then you could try to adapt protocols for isolation of plant RNA and DNA that are developed to remove polyphenols and other contaminants or prevent oxidations.
Examples: PVP (polyvinylpyrrolidone) (remove polyphenols), beta-mercaptoethanol (reduce oxidation).
I am attaching one protocol as an example, but you can also find several protocols for RNA extraction of plants.
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Hello,
I work on serum samples and I'm in a big delimma regarding which primers to choose since I don't have a gene of interest because my purpose is to screen and identify a specific gene.
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Hi! Do you mean to say that you want to do an exploratory study to identify candidate biomarkers from serum? If yes, simple PCR will not be able to identify new genes of interest. You must do some high throughput experiments like RNA profiling or exome sequencing or similar experiments with patient samples and control samples and compare the two in order to identify abnormalities in genetic sequence or gene expression in the disease condition.
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Hi all,
In our lab we have a lot of experience extracting RNA using Trizol.
I know (and have done this) that after taking the top phase and adding isopropanol you can leave the sample in -20c to enhance yield.
I am preparing to extract RNA from critical samples for an RNA seq experiment and I have 2 questions:
1. Does someone have a reference to the fact that the RNA doesn't degrade in these conditions at -20c? I couldn't find one.
2. I also want to add glycogen as a carrier- does anyone know if the addition of glycogen will interrupt the RNA seq protocol in someway?
Thanks!
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Hi Gidi,
just been through lots of RNA preps (Trizol) myself for RNA-seq and we have been using isopropanol precipitation in a 4C cooled centrifuge, occasionally storing samples overnight at -20C but never any longer. These two steps seem to be working equally well but better than spinning at RT.
You can read these articles for some references:
For the second part of your question you can read;
Lots of people do it and does not affect RNA-seq.
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I have read somewhere that these should be only taken as a frame of reference? I am assuming that my samples with a 260/280 ratio of ~1.5 are too low, but when I run it on a 1% agarose gel, the sample appears indistinguishable from my high quality samples that have 260/280 ratios of ~2. Does anyone have any suggestions on the cause for this discrepancy?
Also, would you consider these samples degraded?
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When you measure the ratio of 260/280 by a nanodrop, it simply gives you an idea of purity of your DNA or RNA sample, but not integrity. On the other hand, when you visualize the gel by staining it with an intercalating dye, you will know the quality of your sample in terms of fragmentation/ degradation.
This is the reason why all your samples look similar on agarose gels.
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Hi,
I needed to prepare water saturated phenol for RNA extraction. I did as following: liquefy crystal phenol at 60oC, then add 100ml of liquefied phenol to beaker and followed by adding equal volume of DEPC-treated water, and mixed it by stir-bar. After waiting about 40min, I see three layers, the lowest is transparent, the more upper (middle) is milky, and the higest is transparent. I pipetted off two the uppers and repeated with second adding of equal volume of the lowest. After mixing and wating about 40 min, I could not see phenol phase. What did I do wrongly ? I think crystal phenol is in good condition.
One more, the water saturated phenol will used for RNA extraction. I did not treat container with DEPC , just autoclaved them. Is it safe for RNA working ?
P/S: I am a beginner, and poor for taking such experiment!
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Acid phenol- To solid phenol add RNase-free water until there is a layer of water on top of the phenol: Heat new bottle (500g) to 65 oC, crack lid. Add 100 ml of RNase-free water. Mix and let cool. Add about 100 mls more of water until a little water remains on top of phenol so that is it completely water saturated. Aliquot into 50 ml tubes and freeze at -20.
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In isolation of DNA from trizol method, after adding the absolute ethanol in the phase left behind (RNA Extraction step) i am not getting the 2 layers to proceed further for DNA extraction and protien extraction from aqueous phase. what possible reason for that?
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I am asking.. after the RNA isolation.. i am doing DNA ISOLATION FROM TRIZOL METHOD .. i have to add 100% ethanol in the organic phase( left behind from the aqueous phase after the addition of chlorofrom) . According to the protocol i should have been getting the 2 layers from which i proceed DNA AND PROTIEN EXTRACTION. but am not getting 2 layers
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I have been trying to extract RNA from mouse skeletal muscle to eventually run real-time PCR. The tissue was frozen with liquid N2 and kept at -80. I have been using Qiagen's RNeasy kit (including the optional DNase digestion). I am not able to get a consistent yield even if I re-run samples. I was able to get around 70-90 ng/ul, but recently I keep getting a low yield (around 35 ng/ul). The integrity is fine as two distinct bands appear on an agarose gel.
I homogenize the samples using the Bullet Blender (4 mins @ 8, 1 min @ 9). I do not see any visible chunks afterward.  Any suggestions of how I can improve the yield?
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For those who may be facing this issue, I found that using a bigger piece of muscle, around 30mg, gives decent RNA yield >500 ng/uL, using the Qiagen RNeasy Plus RNA extraction Kit, so there was no need to purchase the Fibrous Tissue kit. Note that I was working with salmon muscle stabilizied in a homemade RNA stabilizing reagent.
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Hello,
For the project I am currently involved in, I am in need for a cheap, non-toxic, pH 7+, non-disruptive buffer for RNA stabilization of bacterial cells (applied to a large environmental sample for subsequent manual cell collection).
RNAlater stabilization reagent is too saline and my cells start floating, so I cannot collect them anymore (the procedure is quite specific).
So I was wondering if anyone could tell me how to prepare RNAprotect from Qiagen. Unfortunately I cannot afford to buy it.
Thanks!
Artur
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Dear Sven. I have addressed Qiagen with the very same question. They replied that RNAprotect Cell Reagent is recommended for "complex" samples, while the Bacteria Reagent is recommended for bacteria only.
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Dear all,
I'm going to perform RNA-Seq with a QIAseq Stranded Total RNA Lib Kit and depletion with Ribominus Transcriptome Isolation Kit/ Ribominus concentration Kit. Does anyone have experience with this kits? Are these kits compatible?
Thanks in advance.
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There need not be be any compatibility between the kits. You can isolate total RNA with any kit and deplete rRNA with any kit available on market.
However, in my experience, RiboMinus from Thermo-Fisher is not at depleting rRNA. THe kit claims 95% efficiency, but the further analysis with bioanalyzer gives different figures and in RNA-seq data. So, in my personal opinion, I would not recommend RiboMinus now.
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Dear all,
I am trying to do an RNA extraction of different bacterial species with RNAeasy mini kit, protokol #4 (lysozyme lysis + proteinase K digestion). Unfortunately, I have a fairly low yield (50-100 ng/mcl) and a high content of gDNA (~20%).
Any ideas how to increase of RNA's yeld? Is it necessary to increase the concentration of lysozyme in the TE buffer ( current 15 mg/ml) or reduce the amount of starting material?
Thanks in advance
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David Farringdon Spencer
, thank you very much for the answer, I work with Yersinia pestis.
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Attached is my total RNA gel picture. Does anyone know that what is the fireball at the bottom of the gel? Is it symbolize degradation? or small RNA?
On the other hand, I did once DNase treatment on my RNA samples (DNase from QIAGEN, off column treatment) then precipitated with Ammonium acetate (left samples are total RNA from bacteria and the right samples are DNase-treated RNA samples shown in the gel picture). From the gel, we can obviously see that genomic DNA is eliminated from the total RNA. However, I obtained bands when I ran PCR to check DNA contamination. Anyone know what is actually happening?
My downstream work will be transcriptome sequencing.
Thank you so much. 
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I think you can try to perform the mRNA capture experiment and then do qpcr to verify several genes. If you can repeat it twice, it means there is no degradation.
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I was extracting miRNA from lung cancer cell(A549) derived exosome by Total Exosome RNA & Protein Isolation Kit from Invitrogen™. I quantified the small RNA concentration with NanoDrop just after isolation. It was showing 1.59ng/ul and 1.29ng/ul respectively for two samples.
I think something is wrong with concentration. Can you please tell me about the normal range of exosomal miRNA concentration?
Is it possible to determine the miRNA concentration from exosome without RT-PCR?
I have to mention that pre-enrichment of exosome had been done by ultracentrifugation for 2 hours at 35000rpm.
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Exosomes have very low RNA amount, what you are getting is about right. Some folks perform qPCR "blindly"- without measuring RNA concentration, esp. when they process very low amount of sample (eg 100 ul serum) and they know that they have an extremely small amount of material, not enough for Qubit or Nanodrop. Others, who have more starting sample- just use maximal possible volume, to ensure they recover decent amount of exosomes and RNA
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Dear Researchers,
I did the qPCR, 20ul, (10 ul TB gree, primer 0.4, template 2ul, Water 7.2), steps, by using TAKARA, I got the following results, as you can see that with 1X, 3X, and 5X dilution, with reference gene ( Histon) Y= yellow variety, P= purple variety, and 1- 9 mean different stages,
the Cq value = 21.6-22.9 Cq erro = (0.01-0.46) ,
which dilution level is ok to use for Targeted genes?
How much cq value and error can be acceptable for reference and targeted gene while doing qPCR?
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Some tips for you:
1) The CV% i accept is 25%. There is no golden rule for this. The CV% must be calculated after 2-Cq transformation. Do not calculate CV% with Cq (Ct) since real time data has a exponential nature.
2) To choose the amplification range with best repeatibility, maintain your samples and standard curve between 15-25 cycles. Do not accept amplification beyond 30 cycles. I recommend this article to full information about real time pcr optimization.
Best,
Jacó
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Hello,
I have problems with RNA extraction from cells. I work with primary neurons and astrocytes cultures, with 1*10^6 cells for each well (more or less, because some cells die).
Actually, I use the Trizol extraction method. With tissue I don't have problems but with cells the 260/280 ratio is horrible, maybe because of Trizol. I don't know. I use the Trizol protocol and I add DNAse in the last step because my probe can react with gDNA.
I use these samples for quantitative PCR, for this reason I need a pure RNA. How can I solve this problem? How can I improve the ratio?
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First I would recommend to keep right ratio of cells to Trizol reagent. For I mln of problematic cells such as neurons I would use minimum 1ml of Trizol. You can use double Trizol method. Isolate RNA by Trizol as usual, then air dried pellet treat with DNAse, then add another 1 ml of Trizol into DNAse/ RNa mixture and isolate RNA again w/o DNAse treatment. Also adding glycogen (see instructions for Trizol) into Trizol itself or into isopropanol helps with higher RNA yields. Make sure you never pick up interphase when collect aqueous phase after chloroform addition- better pick up less aqueous phase, then get DNA/proteins contamination. Also combination of Trizol/columns method will help a lot to get pure RNA. After addition of chloroform and collecting aqueous phase you need to add equal volume of 70% (important!, not 100%) ethanol to aqueaus phase and put mixture on RNeasy columns, then go through steps written in the kits protocol. Also if you have too many dead cells your A260/280 ratio will be much lower compare to healthy (>90%) viability cultures. So you can use Ficoll for example or dead-cell removal magnetic kits to get rid of dead cells. Good luck!
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I'm working on extraction RNA from S. cerevisiae and no matter what I do, my 260/280 looks like this. I'm not asking about the 260/280 itself, because I'm thinking my cells aren't lysing completely, among other things that it could be. My question is how I get rid of the phenol contamination (left shoulder)? Yes, I use chloroform every time. I've tried using columns, not using columns, extra EtOH washes, etc. I don't ever come close to disrupting the interphase when I'm removing the RNA aqueous layer. I don't understand why I can't get rid of that Trizol. Thanks in advance.
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Hi, there are kits for RNA cleanup (polyA or total). I agree with, Adán Valenzuela Castillo but if you have a plan to purify RNA in the future you may consider my idea too.
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We want to extract RNA from small number of oocytes for RNA-seq. I am afraid that I lose RNA with column extractio. I am thinking about using Tirzol. Any idea?
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Hi!
I used Trizol and similar reagents many times to extract RNA from small samples (about 2 mg of tissue). Always cleaning at least twice with 75% ethanol before elution. After total RNA extraction, I usually purify the RNA with the RNEasy MinElute CleanUp (Qiagen) kit. I had some sample loss, but nothing to be afraid of and enough to make RNAseq. If you use this approach, clean the columns twice with the RPE buffer.
The other alternative would be to use the appropriate kit suggested by the colleagues above, or RNeasy Plus Micro Kit (Qiagen).
Best Regards!
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Hi there.
I want to extract separately cytoplasmic and nuclear RNA fraction from leukemic cell line by Trizol. Most of protocols that I saw suggests using a buffer with NP-40 or Triton X-100 for cell lysis. I have a little bit of NP-40, but large amount of Tween-20. May such detergent be suitable for such purpose? If yes, how I can adjust the concentration for extraction?
Thanks in adwance for help!
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It will work, but you may consider performing a titration experiment to optimize the concentration of Tween-20 for your specific needs. It's difficult to pinpoint an exact conversion, as all applications differ. When I swap detergents in this manner, I usually start with a titration of 0.25, 0.5, 1, 1.5, 2x the recommended recipe, then perform downstream analysis to hone in on the best formulation.
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Hi!
We are doing studies about tissue engineering using hybrid scaffold composed of PCL and collagen. However get stuck in RNA extraction for gene expression analysis. Some study suggest using the TissueLysing System (mechanical vibration), yet we don't have the device. Other studies suggest using TRIzol™ Reagent , yet we found TriZol also dissolve the PCL scaffold as well. Is there any other options or reagent kits that can help releasing the cell from the collagen in the hybrid matrix?
Thanks a lot!
Best wishes!
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Actually you can use TRIZOL for RNA extraction from PCL scaffolds. Once the scaffold is dissolved so you are sure that your cells are being lysed too. To see if there is any interference with PCL scaffold and RNA extraction and quantification, I suggest to use a negative control consisting of a normal RNA extraction for just PCL scaffold without cells then subtracting the obtained results from those obtained with cells.
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Dear all,
The fecal samples contain plenty of complex biologically active substance, is there any efficient protocol that increase the percentage of microbial RNA in the extraction of RNA from feces? Additionally, rRNA deleting is also the feature of microbial RNA isolation.
Could anyone please give me some suggestions or reference? Thank you in advance.
Kind regards,
Yang
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MagMax microbiome kit efficiently recovers both DNA + RNA from fecal and many other sample types
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Does anyone know a good protocol to isolate RNA from cucurbitaceous seeds? We are trying to detect viral RNA in seeds by using the silica RNA extraction method but we cannot see any RNA when the samples are electrophoresed in 1,5% agarose gel. Any ideas?
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I recently harvested some bile ducts from mice and need to isolate RNA from them for RT-PCR. I have isolated RNA from mouse liver before with Trizol and the Qiagen Kit but never bile ducts. Would I need to do anything different for this tissue type?
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Perfect. Thank you.
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I'd like to compare the nuclear from the total RNA fraction, especially for small RNA. Is there any good protocol? How to be sure about the integrity and quality of the RNA?
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Because nucleus and cytoplasm contain different amounts of RNA. If you use the same amount of RNA in your workflow, you miss this difference! I now also recommend to add spike-in RNA to the lysates to control for RNA purification efficiency and normalization.
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Hi!
I am trying to extract RNA from apple fruit. At the moment I tried with Trizol but extraction yield and purification steps were insufficient. I am a bit confused: should we use the CTAB method or a plant RNA extraction KIT? The objective is to use the RNA for RT-PCR, and from other works, there is a possible contamination using CTAB method.
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Dear Ana Luisa
Yes, apple fruit is not the easiest tissue to extract RNA, but it is possible. I, first, would try so-called LiCl-based method. There are several modifications of this methods, some use phenol, some, I think, - don't. You maybe will need to try several of them, but since there are enough phenolic compounds in apples, maybe it is better to start without phenol modification.
Also, make sure that You homogenized Your tissue very well in liquid N2, and use RNAse free components and tools.
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I collect bird blood samples in the field, where I can't snap-freeze, so I use RNAlater. Recently, I've heard RNAlater is not recommended for blood. Does anyone also use RNAlater in the field, or an alternative?
We've also had problems with RNA extraction in the past, does anyone have experience in extracting RNA from avian blood?
Thanks!
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We use a 1:10 blood to RNAlater dilution for bird blood. This preservation dilution yields more and better quality RNA than RNAlater at 1:5 and DNA/RNAshield at 1:2 and 1:3. We're writing up a methods papers right now and if you'd like to chat more about it, I can put you in touch with my post-doc!
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I want to kno the reason why i got high value for 260/289 ratio and 260/230 ratio
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Blake Balcomb and Md Abdur Rahim true
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Dear all
I have tried 1ml Trizol to extract total RNA from crabs gills. The weight of most of my samples is + -0,03 g. We have applied this protocol for hepatopancreas samples and the results were very good. However, for gills, the efficience of RNA extraction was ok for less than 25% of samples. We are using petles for microtubes (RNAse free).
Any information you can provide me would be greatly appreciate.
Cheers
Isabella
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I have never extracted RNA from crab gills either, but it could be how well you're breaking up your tissue in question. One way to get better RNA yields through more durable tissue is by freezing it down with liquid N2, grinding it up, and then resuspend in 1ml TRIzol. However, with this method sterile technique needs to be a constant thought.
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Hi!
I have mRNA after in vitro transcription. Nanodrop shows conc of 4150 ng/ul, (measured at 10x dilution, 415ng/ul), but Qubit and Tapestation show 10 times lower concentrations. The gel from Tapestation shows very weak band. I do phenol chloroform purification after IVT which is suppossed to remove most of the free nucleotides. The A260/280 ratio is 2,05 and A260/230 ratio is 2,34. I have no idea where does this difference come from. I tried different Nanodrops and different Qubits...
Maybe you can help?
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qubit reading are also affected by salts and other components present in the sample. Few chemicals are validated and published in qubit dye manual too. same is true for spectro, it will give reading for anything, having absorbance spectra in that range, we are measuring
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Dear all
i plan to extract the mitochondrial DNA and RNA from plant tissues to identify and analyse the process of RNA editing.
in this case i have been searching online but there seems to be more protocols with a numerous reagents and steps.
i was wondering if any company like Qiagen, Thermofisher or.... offer any kit which is fast and reliable so that i can buy.
regards
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I am looking for methods (or kit) for mitochondrial RNA isolation. I need to go for RNA-seq. Could you suggest me in this regard.
Thanks
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Hi! I am trying to isolate RNA (for qPCR) from low numbers of sorted astrocytes (through FACS), using Qiagen RNeasy Microkit as I have samples ranging between 500 cells to 30k cells (due to different sizes of the starting tissue). So far, I obtained very low yields (< 10 ng/uL) and really bad 260/280 and 260/230 ratios at Nanodrop from all the samples.
I usually sort cells, keep them in ice, pellet them, remove surnatant and add 350 uL RLT buffer, then I either freeze or continue immediately with the protocol. I homogenize with an insuline needle, but I don't know if this might be the problem.
- Does anybody have tips to carry out the protocol in the most accurate way?
-Does anybody recommend using Trizol instead for these low numbers of cells?
I red that some people sort in RLT. Can someone comment on the usefulness of doing so?And if you do sort in RLT, how much RLT you put in the collector tube, considering that while sorting you also collect the sorting medium?
Any suggestion of any kind would be bery much appreciated!
Thank you all.
… 
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Hello-- don't be discouraged by your poor RNA results (especially with the nano drop). I work with FACS sorted microglia (also in low numbers) and also had poor results on the nanodrop of RNA extracted via the RNAeasy kit.
Using the PicoPure RNA extraction kit (https://www.thermofisher.com/order/catalog/product/KIT0204), which is meant for low levels of RNA (which seems to be your case) I similarly had poor nano drop results, but the Agilent Bioanalyzer analysis indicated high quality RNA extraction (RIN above 9). I would encourage you to try a different extraction method. I sort directly into (RNAse and DNAse free BSA (1%) with RNAse inhibitors). See 10x genomics protocol for the "Rehydration Buffer": https://assets.ctfassets.net/an68im79xiti/1VpFPpPu8UKu8EeCOYGIAa/c79aa5c0fbb970f6d879c37b12b15874/CG000136_SamplePrepDemonstratedProtocol_-_MethanolFixation_RevB.pdf
Often the levels of RNA are so low that the nano drop cannot accurately detect it.
I hope this helps!
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I've been trying to extract rna from this particular yeast strain By611 and I could only get one band out of it. I am planning to run q-pcr after this step. Below I am attaching the picture of my gel. Would like to know what could be the possible reasons I am not getting the bands.
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Kavilasha Venugopal its good method we use it in our lab . be sure to use RNase DNase free tubes and to use DEPC-treated water to dissolve the RNA pellet ,also the 70% ethanol to be prepared using DEPC water .
Good luck
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I am looking to perform RNA extraction on whole blood samples currently in Paxgene tubes using a Qiagen Blood RNA Extraction kit designed for use with Paxgene tubes. The protocol calls for 2 steps that require a shaker-incubator at 55-65C and 400-1400 rpm for only 5-10 minutes. We have asked other neighboring labs to use theirs but it is difficult to find a time that works for everyone, and shaker-incubators are very expensive. We do have an incubator, it just doesn't shake, so I'm wondering if we can just leave them to incubate at the required temp and then every minute or so take them out and manually shake them for a bit then put them back in.
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Hi Julien,
First of all I didn't use this kit before. We use K2Edta tubes for isolation and do RNA isolation with trizol in our lab. And we do manual shaking on some steps, just like yours and we had no problems.
I think you can shake your samples manually. I don't think you will have problem.
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I injected carrageenan on mice hindpaw to evaluate neuropathic pain and inflammatory response. After RNA extraction (by Trizol), reverse transcription and real time PCR, only the control group (saline injected, without carrageenan) had positive amplification.
Since carrageenan is interfering with my reactions, is there a safe way to get rid of it?
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Dear Lucas,
I know my answer comes quite late. I have seen that RNA extraction is performed in Chondrus crispus with RNeasy Plant Mini Kit from QUIAGEN (Kowalczyk N, Rousvoal S, Herve C, Boyen C, Collen J (2014) RT-qPCR Normalization Genes in the Red Alga Chondrus crispus. PLoS ONE 9(2): e86574. doi:10.1371/journal.pone.0086574). As this is the algae from which carrageenan is mainly extracted, it may worth to give it a try. I will like to ask you if you managed to find any solution for cleaning up your RNA from carrageenan as I may be experiencing a similar issue.
Kind regards,
Adrian
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Our problem is relative to the disruption methods, we tried different methods like, sonication, grinding with beats, and liquid nitrogen, although we get some results we did not obtain an high RIN yet.
Someone have a different protocol? 
Thanks in advance. 
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Dear Mr. Antonio Ida
Would you mind to share your protocol in your laboratory for RNA extraction with trizol?
Thank you so much
Regards,
Feisal
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I am trying to perform trizol RNA extraction on mouse skin samples. After addition of chloroform for phase separation, the top aqueous layer remained very pink - I think from Trizol contamination. However, we progressed to adding isopropanol to precipitate the RNA. No pellet was seen, even though the same batch on a previous day showed good concentration and purity of RNA extraction. 
Is it possible to repeat the chloroform step again at this point after isopropanol addition to try to further remove trizol contamination? The samples still look very pink. 
Any other suggestions would be greatly appreciated.
Thank you very much. 
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you should use 70% ethanol to purify and precipitate the RNA
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I want to redo a RNA extraction from the E.coli cells and I store the liquid culture (in TB) for two days. I am not sure whether I can still use them or I need to regrow some cells.
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Assuming you are planning to look at mRNA, then you need fresh cells. Most messenger RNA in E. coli will turn over quite quickly.
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I am using QIAGEN RNeasy Mini kit to extract viral RNA, I have seen use of this kit in many studies. To optimize the procedure, I start with cell culture virus isolate (I will work with environmental samples) and have been using betamercaptoethanol. However I keep getting low RNA concentrations (0.9 - 1 ng/ul).
I cannot use Viral RNA Extraction kit, the only thing I have RNeasy mini kit
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Your welcome.
It is good to hear.
Good luck
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we use trazol kit for extrcting RNA from sputum ;although we change our extraction kit we faced low 260/280 ratio of nanodrop.(1.5)
according to well-known contamination of sputum (carbohydrate);
what is the best solution for obviating contamination with less RNA destruction?
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Dear Ahmad,
I've worked with RNA from plant cell cultures, with long amounts of carbohydrates and polyphenols, which produce interference with RNA extraction. I recommend you that use some sequestring agents in your extraction buffer, like PVPP (Polyvinil(poly)pirrolydone at 2%. PVPP is water insoluble, but can be easily removed from your RNA sample by centrifugation. Other option is carry out the RNA precipitation in two differents steps, one with 2-propanol and Sodium acetate 3M pH5.2, and other with Lithium Chloride 10M. (Morante-Carriel et al., 2014).
Good luck!
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Hello everyone, this question has confused me for a while.
I am trying to extract RNA from mice brain for RNA sequencing.
However, I just noticed that the original protocol I followed recommend to use glass pestle A and B, like homogenize the sample first with pestle A and then with pestle B for 15-50 times.
For a long time, I homogenized the mice brain cortex by plastic grinder in 1.5 ml tube with homogenization buffer first and then pipetting add up to 1 ml. After centrifuging, I collect the supernatant for RNA extraction by Qiagen RNeasy Micro Kit. But the RIN value is always around 6.
So, I started to wonder whether I used the wrong tool or method to homogenize the brain tissue?
Because of the budget, I can not afford the glass pestles.
I just consider using the stainless grinder.
That's why I want to know the difference between glass pestles and stainless grinder. Will it be the key for the RNA quality?
Thank you.
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Hello, Jack.
I do think you will have even better results with stainless steel beads in microfuge tubes like eppendorf safe lock tubes. Systems like MM400 homogenizer (tissuelyzer from QIAGEN) helps to keep samples under low temperature. This is (in my humble opinion) the most important step to achieve the best results in RNA extraction. Of course, minimum time from sampling to extraction and cDNA synthesis.
I did use RNALater but, please, read instructions because, sometimes, people think they should keep samples fromzem with the solution, which is NOT the case. Ok?
I hope I shed some light on your question.
Best of luck.
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I want to extract total RNA from pichia pastoris to evaluate the yeast transcriptome using RNA-Seq.
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Is that absolutely necessary? look at this ref they simply extracted RNA using RNAeasy kit from treated and untreated condition.
Best wishes,
Hanna
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Protocol content (NEB DNAse I reaction Protocol) :
RNA: 10ug
DNAse reaction buffer 1X 10ul
DNAse (Rnase free) 1ul
Nuclease free water 100 ul
My question is; if I use this protocol after isolating total RNA, I dilute the RNA ratio.
If I use it during RNA isolation, I am not yet getting RNA according to the protocol.
At what stage should I use?
Thank you!
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If you use post extraction digestion then you just dilute for the extraction protocol. However, since you will need to purify again after the digestion to remove the DNAse you will reconcentrate the sample to the original concentration. For example if you eluted with 30ul for the original extraction then you can digest in a total volume of 100ul (you diluted here). Then after the digestion you do phenol chloroform and afterwards resuspend in 30ul again therefore the only amounts that you lose is what is lost during the precipitation. I suggest is that you follow the on column DNAse digestion to circumvent going through all of this miss it is written in the kit's appendix D and I will copy it here for reference (just be sure to use the DNAse set that is from qiagen since on column digestion is only compatible with their set ) essentially you split the RW1 wash step into two first you wash with a smaller volume of RW1 then incubate the column with dnaseI suspended in buffer RDD. Then you wash away with the seconds half of RW1 buffer (to remove the dnase I)
Full protocol (copied from RNAeasy kit):
Appendix D: Optional On-Column DNase Digestion
with the RNase-Free DNase Set
The RNase-Free DNase Set (cat. no. 79254) provides efficient on-column digestion of
DNA during RNA purification. The DNase is efficiently removed in subsequent wash
steps.
Note: Standard DNase buffers are not compatible with on-column DNase digestion.
Use of other buffers may affect the binding of RNA to the RNeasy membrane, reducing
RNA yield and integrity.
Lysis and homogenization of the sample and binding of RNA to the RNeasy membrane
are performed according to the standard protocols. After washing with a reduced
volume of Buffer RW1, the RNA is treated with DNase I while bound to the RNeasy
membrane. The DNase I is removed by a second wash with Buffer RW1. Washing with
Buffer RPE and elution of RNA are then performed according to the standard protocols.
Important points before starting
Generally, DNase digestion is not required since RNeasy technology efficiently
removes most of the DNA without DNase treatment. However, further DNA
removal may be necessary for certain RNA applications that are sensitive to very
small amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundant target).
DNA can also be removed by a DNase digestion following RNA purification.
Do not vortex the reconstituted DNase I. DNase I is especially sensitive to physical
denaturation. Mixing should only be carried out by gently inverting the tube.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
† To make a saturated solution, add solid bromophenol blue to distilled water. Mix and continue to add more
bromophenol blue until no more will dissolve. Centrifuge to pellet the undissolved powder, and carefully
pipet the saturated supernatant.
RNeasy Mini Handbook 06/2012 67
Things to do before starting
Prepare DNase I stock solution before using the RNase-Free DNase Set for the first
time. Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNasefree
water provided. To avoid loss of DNase I, do not open the vial. Inject RNasefree
water into the vial using an RNase-free needle and syringe. Mix gently by
inverting the vial. Do not vortex.
For long-term storage of DNase I, remove the stock solution from the glass vial,
divide it into single-use aliquots, and store at –20°C for up to 9 months. Thawed
aliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots
after thawing.
Procedure
Prepare and load samples onto the RNeasy spin column as indicated in the individual
protocols. Instead of performing the first wash step, follow steps D1–D4 below.
D1. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and
centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane.
Discard the flow-through.*
Reuse the collection tube in step D4.
D2. Add 10 µl DNase I stock solution (see above) to 70 µl Buffer RDD. Mix by gently
inverting the tube, and centrifuge briefly to collect residual liquid from the sides of
the tube.
Buffer RDD is supplied with the RNase-Free DNase Set.
Note: DNase I is especially sensitive to physical denaturation. Mixing should only
be carried out by gently inverting the tube. Do not vortex.
D3. Add the DNase I incubation mix (80 µl) directly to the RNeasy spin column
membrane, and place on the benchtop (20–30°C) for 15 min.
Note: Be sure to add the DNase I incubation mix directly to the RNeasy spin
column membrane. DNase digestion will be incomplete if part of the mix sticks to
the walls or the O-ring of the spin column.
D4. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and
centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.*
Continue with the first Buffer RPE wash step in the relevant protocol.
Note: In most of the protocols, the immediately following Buffer RW1 wash step is
skipped (as indicated in the protocol). Continue with the first Buffer RPE wash step.
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I am using stepone PLus machine and in excel sheet attached to my plate . i found effiency mentioned 1? what does this mean? iam quiet new to the device
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Yes, relative quantification in qPCR is calculated using the delta-delta Ct method. The efficiency is how closely your primers match the desired rate of product formation. To publish your results, your primer efficiency must be 95-105%.
Here's a publication to get you familiar with the MIQE guidelines to publish your qPCR data:
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I will be treating heparinized whole blood with a small molecule for various timepoints and then extracting RNA for qPCR.
Do people recommend incubations in tubes or plates/dishes?  5% CO2/37C or only 37C?  I am set on the RNA extraction part, but fairly inexperienced with the prior steps, so any hints or tips would be well appreciated.
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Hello,
I would be so thankful if someone could lend us a hand with an issue related to RNA purification.
I'm using the QIAGEN RNeasy Plus Mini Kit for RNA extraction from brain tissue and adipose tissue. I have a set of hipothalami with 1.0ug/ul-1.4ug/ul, and a RIN of 9.4-9.8. But in another set, I got 0.1ug/ul-0.4ug/ul. We should have the RIN of this second set in a couple of days, but I can't get good sleep with this problem.
The manipulation conditions were the same. As soon as the tissue is extracted, it's freezed. The tube contains ceramic beans for later homogeneization with Paracellys tissuelyzer. I always use the same program for tissue disruption. Then I just follow the QIAGEN protocol, with very few adjustments, which I maintain for all the samples. I elute the RNA using 50ul of PCR grade water, and I always incubate the samples 10min before elution at RT.
We need concentrations of at least 0.4ug/ul for qPCR.
Can these concentration difference be due to individual differences? Or is there a flaw in the procedure, significant enough to result in these differences?
Your help and suggestions are much appreciated. Thank you very much in advance!
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I am currently trying to understand the RNA extraction procedure. We used a plant RNA extraction kit (the E.Z.N.A plant RNA extraction kit from omega bio-tek) that involved cell lysis with a homogenizer column, addition of ethanol and then an RNA mini column that binds the RNA, and then we eluted the RNA using DEPC water.
What I want to know is how the RNA mini column only binds to RNA and not DNA? Is it something to do with the buffers having DNases so the DNA is degraded, or the column deferentially binds to RNA over DNA? Or does this depend on the kit used as to how only RNA is extracted?
Any insight would be extremely appreciated.
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Hello,
Generally, RNA extraction kit contain DNA removal step such as treatment of DNase or acidic phenol (e.g. trizol).
In acid condition, negative charge on phosphate backbone of DNA and RNA is neutralized with H+. DNA becomes uncharged, but RNA remains water soluble because it was exposed nitrogenous bases on purine pyrimidine (RNA is single-stranded). Therefore, acidic phenol method can separate those by treating additional solvent (e.q. chloroform)
In silica base method,
even though silica is negative charge, in high salt (chaotropic salt), the cation play a bridge between silica and phosphate backbone on DNA or RNA. (i.e. charge binding)
i think that silica column can't separate DNA and RNA, perfectly..
some time ago, i extracted high copies of DNA viral genome (for enteric adenovirus) from environment samples with viral RNA mini kit (silica based method).
Good luck.
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RNA extraction from laser microdissected FFPE tissues is markedly enhanced by sonication (paper below). However, I could only find a kit and protocol for Covaris sonicators. We only have a single-tube Covaris instrument, while we also have a multi-tube Bioruptor. Has anyone tried to use Bioruptor for LCM samples from FFPE tissues?
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sonication is tricky, and FFPE samples are very challenging (RNA is degraded and cross-linked, and trapped in this matrix). for RNA recovery from FFPE, follow these MagMax and RecoverAll protocols: https://www.thermofisher.com/order/catalog/product/A31881
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I had to perform RNA extraction from the mice skin sample (shaved and hair removed tissue sample of about 1*1 cm square). For that I pulverized the tissue in liquid nitrogen pre-chilled mortar and pistle and extracted RNA with Trisol reagent (Trisol-chloroform-isopropanol method), general method for RNA extraction. While quantification, I got sufficient RNA with good quality (I mean 260:280 ration is >1.9 -2 and 260:230 is >1.5 in each sample. I made cDNA (using 1 microgram of this RNA) and performed the qRT-PCR. Surprisingly some sample Ct value is not detected and other also have the quite high variation in Ct value. I tried with different house keeping genes (HKG) (actin, GAPDH, CycloQ, 18s etc In HKG also there is variations in Ct value in among normal mouse (eg. Ct value ranges from 14-19 form normal group). However, according to in many researches there was no problem with the RNA extraction methods in my case, I am unanswered that where is the wrong gone? For other tissues, such as brain, I got perfect results (almost same Ct value for normal group with HKG). Could you please suggest me where is the problem in my case? I will be happy to get the answer from the researcher who did the RNA -qRT-PCR in skin sample (experienced one).
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Thank you for Dr. Bouke and Kartiki for the suggestion. Dr. Bouke, I think pulverizing is sufficient, because the yield was high enough that is, ~800 ng/microliter. At the moment I am not separating the dermis and epidermis. But haven't use bead-beater yet. Is there any other method beside this?
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I need to extract miRNA from blood plasma in glioma patients. I did it by GeneAll kit but RNA yield is low. Who can help me to improve the yield by using this kit or to find a manual method that works well.
Best Regards
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Dear all, 
We’re currently extracting total RNA from gammaridean amphipods (Gammarus pulex). Animals were freshly collected, directly frozen at -80°C.
Trying different RNA extraction protocols (Qiagen, Trizol+Qiagen) we always end up seeing no obvious 28S band. First we thought it is due to the denaturing step prior to loading onto the Fragment Analyzer (known issue). But omitting this heat denaturing step does not lead to a change - the picture remains (see attachment for examples). Any ideas? We also gave the samples to a different lab - extraction results the same. There seem to be sometimes two only marginally different peaks at the 18S band position and we suspect that something other than heat is leading to the dissociation of the two 28S subunits possible. If these are of same lengths as the shorter 18S fragment can this explain the pattern… So non-model organisms experts: Can someone help us with this puzzle? Best Florian and Team
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Hi Florian,
I know this phenomenon also from my work with protozoans. The LSU rRNA dissociates into 2 (sometimes ~equally sized) fragments due to a "labile" or "hidden break" region that undergoes a fairly rapid processing in vivo under denaturing conditions. But in my experience this also happens during non-denaturing gel-electropheresis or Bioanalyzer runs and might have already happen during the extraction. This "hidden break" is also known from insects, trematodes and fish. Maybe this causes your missing 28S band as well. A very gentle RNA extraction may could help. Ogino et al. 1990 (J Mol Evol, 30:509-513) or ( ) might be interesting to read.
Best,
Sascha
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Hi All,
I was wondering if anyone had recommendations , insights, comments, etc. on what they think is the best RNA extraction protocol for small tissues. By small, I mean the brain of a tadpole that is ~3 mm cubed (the brain itself). We have a protocol that can yield ~8-10 ng/uL. However, I was wondering if others have had experience with similar sized tissues and if they have any recommendations. Any insights would be greatly appreciated!
Thanks,
Nick
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Thanks for the answer and recommendations, Tania! They are really helpful.
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iam extracting RNA from brain tissues that are stored in -80 .should i get them all at same time from -80 which starting to homogenize some of them on ice or get few sames out of the -80 everynow and then during the process? to ensure treating all samples the same ? any advices ,papers will be beneficial thanks in advance
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You could also keep all your samples in dry ice while extracting RNA from a small number of samples. It's important not to let them thaw.
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I used RNAeasy Plus minikit but I didn't get any RNA. I extracted RNA in parallel from Hela cells and I obtained the expected yield. The lysis buffer was added directly on Transwell filters in 6 well plates after removal of the growth medium. After extensive pipetting at RT the samples were transferrred to 1,5 ml reaction tubes and immediately processed according to manufacturer protocol with addition of the QIAshredder step.
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Exactly, I added 1,5 X volume of alcool. An higher EtOH:sample ratio before loading on columns is suggested by different brands of RNA purification kits depending on the tipe of sample. For me it worked but I still think that the main problem with Caco2 differentiated on filters is cell lysis so I preferred to use a phenol based extraction buffer.
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I tried to extract plant virus (RNA genome) from ginger leaves but there are a lot of inhibitors when I come to cDNA and RT-PCR sometimes. I want to get expertise advice from those who worked with the plant.
Thank you.
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Dear Muhammad,
we also met same problem in our study. You can use charcoal during extraction of leaves for reducing effect of inhibitors. I am sending web connection of our study as follows:
I think and hope it will be helpful for you. Good luck.
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I am extracting RNA from chytrid fungi using an Ambion Purelink RNA mini kit and quantifying the RNA yield using a Qubit 3.0
The majority of yields have been <1ng/ul.
Do you have a protocol for Extracting RNA from chytrid?
I successfully extracted DNA from chytrid using a the genejet columns but seem to be struggling to get RNA. I am using RNAse Zap bewteen steps and anytime a tube is opened and changing and treating gloves with RNAse Zap frequently.
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Can you explain your lysis step? This is very important and crucial for the yield of RNA
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I'm using the RNAzol for extracting total RNA from Gram-positive bacteria that have been harvested in the mid-exponential growth phase. However, we observed bacteria growth is extremely slow for unknown reasons. Could it affect the quality of extracted total RNA?
When verifying the RNA quality by the Bioanalyzer (Agilent) we observe the 23S rRNA peaks are lower than 16S rRNR (RIN>9). Sometimes 23S RNA peak is extremely low although degradation products between theoretical 16S and 23S rRNA peaks are not seen (see figure).
Is it degradation? Or are there other possibilities?
How can I avoid this?
I would really appreciate any advice to cope with this situation.
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Dear Ramona
Yes it can but in my experience extraction from stored RNA will generally result in that lower 23s peak
To answer your question: no you will absolutely lose mRNA from your sample if the ratio of your rRNA changes; the 2 are completely independent
To reiterate your RNA - for stored RNA - is of high quality as your signal to noise is very good and your RNA is devoid if degraded products; hence your high RIN number
The additional prominent low Mr peak However does concern me: as somebody who has examined Agilent traces for 20 years and worked with RNA for 30 years in effect what I see when I look at your trace is relatively clean undegraded RNA but contaminated with GITC. That being so you do need to remove that peak (contamibation) by 're ppt your RNA as explained and then desalt with 70% ethanol x 2
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We are culturing cells inside a 3D PLGA electrospun scaffold. Currently, we are using Qiazol to isolate the RNA, and we found that the Qiazol dissolves the PLGA scaffold upon contact. And we also found that the RNA subsequently extracted via mini Qiagen kit in this manner was degraded when compared to that obtained from 2D controls. Has anyone successfully extracted RNA from a PLGA scaffold?
We also tried to trypsinize the cells down from the scaffold, however the cell yield is very low leading to very low concentrations of extracted RNA.
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in your RNA isolat kit you have lysis buffer, use that to lyse your cells from inside of your scaffold. Be quick, RNA is not stable.
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I am trying to adapt a DNA extraction protocol to allow me to extract both RNA and DNA from the same sample. I plan to put the sample through Qiagen Allprep DNA/RNA mini kit, but I am not sure at what step I lose the RNA.
SDS/CTAB cleanup
a. Add 10 μl SDS (10%) + 1 μl proteinase K (10 mg/ml stock) to each tube and incubate at 56 °C for 20 min. At this point, pre-incubate CTAB/NaCl solution at 65 °C.
b. Add 35 μl NaCl (5 M) + 28.1 μl CTAB/NaCl (2.5%). Pulse vortex. Incubate at 65 °C for 10 min then perform a quick spin.
c. Add 200 μl phenol:chloroform:isoamyl alcohol (25:24:1) pH 8.0. Pulse vortex. Centrifuge 8,000 × g for 5 min at RT.
d. Collect the aqueous fraction. Add 200 μl chloroform. Pulse vortex for 3–5 sec. Centrifuge 8,000 × g for 5 min at RT.
e. Collect the aqueous fraction. This is final Virus Nucleic Acid.
The final viral nucleic acid goes through the Qiagen DNeasy Blood and Tissue kit.
I am not sure if the final nucleic acid contains RNA. If it does not contain RNA, I would like to know at which step I should put my sample through the kit.
I read protocols using CTAB to extract RNA as well as DNA, but I am not so sure about phenol:chloroform:isoamyl alcohol or plain chloroform. I read a similar protocol for extracting RNA that used CTAB and phenol:chloroform:isoamyl alcohol, but they replaced the chloroform with isopropanol and centrifuged, collecting the pellet as final RNA.
If someone could help me sort this out, it would be great!
FYI, this is part of a protocol to enrich for viral particles and extract the nucleic acid from stool with the least amount of human or bacterial contamination.
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As others have said, your high pH phenol:chloroform:isoamyl alcohol (25:24:1) will not separate RNA and DNA - and neither will your DNeasy kit. Your best bet is Trizol which retains DNA in the organic phase that can later be isolated.
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Hi everyone,
I have taken blood samples from fish, I plan to use these samples for transcriptome analysis (RNA-seq). Unfortunately due to cost I was not able to purchase PAXgene or Tempus tubes.
so far i have taken 1ml of whole blood from each fish.
I have centrifuged the samples and taken off the plasma. to the remaining I have added 1.5ml of RNAlater solution, in the hope of stabilising the cells. I flash froze the solution and it is currently stored at -80.
I require a RIN number of 7 +, and am looking for recommendations of what kit / method I should use, or is it to late to expect a good quality yield.
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Just came across to this question and I'm curious about what kit/method did you finally use for extracting RNA. I have the same issue with my samples; they are blood (plasma taken out) stored in RNAlater... So far I have not find an accurate protocol for that...
Maybe you could share your experience? I would really appreciate it!
Thanks.
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I have come across a method for RNA Extraction named RNASwift by the authors (RNASwift: a rapid, versatile RNA extraction method free from phenol and chloroform, but Nwokeoji et AL., 2016).
This method really interests me as only uses SDS 4% and Sodium chloride to extract RNA. But I am skeptical of its efficacy. The article states that SDS would denature proteins, but I don't understand why it would remove DNA as well, and why RNA will remain in the extract.
Has anyone attempted this method? If so, what is your opinion?
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Good day! It's me again.
After isolating "something" which absorbs at 260 nm using the RNASwift protocol, I treated it with Turbo DNase, this resulted in a slight decrease in concentration but the majority remained. Then I performed agarose electrophoresis, denaturing RNA with formamide (50% final concentration), and found that although the obtained RNA was very clean, it was degraded at some extent.
Comparing with RNA obtained using TRIzol I found that although a little bit contaminated with guanidine salts and some proteins (because of 260/280 and 260/230 ratios; ie. 1,8 and >1.0) it conserved integrity.
I think RNASwift is an excelent alternative, but I still need to optimize it for my purpouses.
I attach a photograph of the gel.
Lanes: 1, DNA ladder; 2 and 3: RNA obtained with TRIzol; 4-6: RNA obtained using RNASwift from 1.0, 0.2 and 1.8 mL of culture.
I would like to know about your experience.
Best regards.
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Hi all,
I have been extracting total RNA from E.coli and B.subtilis cells grown to OD600 0.5-1.0 for two years now using the protocol detailed below. Things that vary from extraction to extraction are 1) the phenol may be freshly prepped every 5-7 extractions because I run out, 2) the 6M NH4OAc pH 5.3 solution is a stock solution that I use every time and ages with each extraction (don't know if that's actually a problem). Recently (in the last month and a half) I have been performing the exact same extraction/digestion procedures and have been getting very little to no ion signal on the MS, even with the major nucleosides (uridine, adenosine). I have run older samples from two months ago along with synthetic nucleoside standards every time alongside the newly extracted low signal RNA samples and the older sample/standard signals are excellent, so I have eliminated the possibility that it is the instrumentation or the digestions (if either of these failed, I would be getting no signal for all samples because I digest stored older RNA and newly extracted RNA at the same time and run them at the same time on the MS). Over the last month and a half nothing has changed except that I have made new 1M NaOAc pH 4.5 saturated Phenol 3 separate times, suspecting the phenol as the culprit after getting no signal the first few times it became an issue. I am at a loss and would love a set of fresh eyes to look over the protocol to see if there are any glaring discrepancies this signal loss can be attributed to! Switching to Trizol reagent is also not an option for this project.
Protocol
1. Resuspend pellet in 10 mL 0.3M NaOAc/10mM EDTA pH 4.5 solution (starting media volume of pellet is 500 mL of culture for 10 mL resuspension)
2. Add 3.5mL of 1M NaOAc pH 4.5 saturated acidic phenol to resuspended pellet and vortex for 60 seconds (pause for 30 seconds) and vortex for 90 seconds
3. Spin at 14,000 rpm for 15 minutes at 25C; keep top layer and transfer to new tube.
4. Add another 3.5 mL of 1M NaOAc pH 4.5 saturated acidic phenol to top layer
5. Vortex for 60 seconds; Spin at 14,000 rpm for 15 minutes at 25C; take top layer and transfer to new tube.
6. Add 0.5g NaCl and 10 mL of a 24:1 Chloroform/Isoamyl alcohol mixture. Vortex for 15 seconds; centrifuge for 3 minutes at 14,000 rpm at 25 C.
7. Take top layer and transfer to a new tube. Fill tube up to 50 mL mark with chilled 100% (200 proof) Ethanol and precipitate at -20C overnight.
8. Centrifuge at 14,000 rpm at 4C for 25 minutes; decant ethanol layer and do not disturb precipitate (RNA is in precipitate).
9 . At 10 mL of 70% ethanol to pellet and vortex for 15 seconds
10. Centrifuge at 14,000 rpm at 4C for 30 minutes; decant ethanol layer and do not disturb precipitate; Allow pellet to dry inverted in hood.
11. Resuspend pellet in Optima water and add to a final [2M] NH4OAc from your 6M NH4OAc (pH 5.3) stock solution (stored at RT) up to 1 mL final volume.
12. Take [RNA] on Nano-drop (260/280 should be around 2.0)
13. Take 60 ug of RNA and heat denature at 100C for 5 minutes
14. Chill on ice for 5 minutes
15. Add 10uL of P1 Nuclease to denatured RNA
16. Add 10uL of 0.1M NH4OAc (pH 5.3) and Optima H2O up to 150uL.
17. Incubate at 50 oC for 4 hours
18. Add 20 uL of 0.1M NH4HCO3 to incubation mixture (Make this solution fresh daily)
19. Add 20uL of BAP Buffer (bacterial alkaline phosphatase)
20. Add 10 uL of BAP enzyme
21. Incubate at 37C for 2 hours
22. Centrifuge at 14,000 rpm for 5 minutes; take supernatant and spike to a final [MeOH:Formic Acid] 2%/0.1% respectively
  1. Tags: RNA Extraction, LC-MS, Bacterial RNA Extraction, Ion Signal,
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This protocol is perfect, but may be you have a contamination in addition the phenol chlorophom better than Trizol ( in my opinion)
regards
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I accidentally put a piece of tissue in a tube containing medium+DMSO to be used for RNA extraction later on. 15 minutes after putting the tube in the -80 I realized that I made this mistake so I washed the tissue with medium and put it in an empty tube before transferring it to the -80 once again. Do you think the RNA got degraded because of the DMSO?
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But if you use frozen cells (in regular freezing medium with 10% of DMSO) for RNA extraction and subsequent RT-PCR/qPCR performance, DMSO won't be an issue, right?
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Hi,
I'm trying to extract RNA from PBMC after culture or stimulation. I'm using a old fashion protocol: lyse of the cell with RNAble, addition of 10% chloroform, collect of the upper aqueous phase, precipitation of the RNA in isopropanol, then 2 washes in EtOH 75%. Let dry the RNA and resuspend RNA in water.
With this protocol I had good ration A260/280 but very bad ratio 230/260.
To improve the ratio, I decided to wash 3 times in EtOH 75% and to perform a final wash in Absolute EtOH (Also to improve the EtOH evaporation and so the drying of the RNA.
I didn't really improved the 260/230 ratio that are actually terribly bad (0,1 to 1,3).
I'm not an expert in RNA extraction but before I extracted RNA from tissue using column kit and had very good ratio.
Now I extract RNA from PBMC, and since the quantity of RNA extracted is very low, my lab use the old fashion protocol to improve the quantity retrieved.
Anyway, I would like to understand what to do to improve the A260/230 ratio.
I have to say also that because the pellet of RNA is barely visible I never eliminate the totality of the washing buffer (EtOH 75 or 100%) to be sure to not discard the RNA, but because I increased the number of washes, I thought it should be ok but apparently not.
Any ideas???
Thank you very much
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With the little experience that I have in RNA isolation, a low 260/230 indicates phenol like contamination. Trizol is a good method to get clean RNA with un-compromised integrity. Moreover, the yield is very high vis-a-vis kits. Take the aqueous layer very carefully trying not to disturn the interface at all. Also, try to reduce the time for RNA precipitation to 25 min, centrifuge and then wash twice with 75% EtOH made in DEPC water preferably. After last wash allow EtOH to vaporize. Don't overdry. Resuspend in DEPC water
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I'll work with fecal samples and I want do a good storage for a good RNA extractions in other occasion, I want to know the ratio of fecal samples and RNAlater for a good storage.
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hope this link is useful
https://www.nature.com › scientific reports › articles
regards
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I would like to isolate total RNA from single cells for qPCR (or even sequencing later on). What protocol or kit should I use to obtain good quality RNA with minimum loss?? I've seen there is a kit "PicoPure® RNA Isolation Kit" but I'd like to know if there are other suggestions before buying such and expensive kit.. 
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try single cell "direct lysis" Cells-to-Ct kit
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I have been trying to isolate RNA from human fat sample without success. I have followed both the trizol protocol from Invitrogen and the Rneasy Lipid Tissue Mini Kit and ended up with concentrations around 5 ~ 40 ng/ul. How do I increase yield and quality of RNA?
Any help is much appreciated. Thanks in Advance!
Samples are obtained from the OR (sample baths in tubes with KRB with 1% BSA on ice), then washed with KRB with 1% BSA(Room temp) to pick out the connective tissues and flash freeze in Liquid nitrogen.
The trizol protocol is as follows:
  1. homogenize/sonicate samples in 1 ml Trizol
  2. spin at 4C on 12000g for 5 mins and remove fat layer on top
  3. incubate for 5 mins > add 200ul chloroform > incubate 2 mins
  4. spin to separate phases > transfer the aqueous layer to new tube
  5. add isopropanol to precipitate RNA >vortex > spin 12000g at 4C
  6. wash pellet in 75% ethanol > vortex >spin 7500g at 4C
  7. dry pellet for 10 mins (proceed if ethanol is less than 20ul)
  8. resuspend pellet in 30ul in RNase free water
  9. incubate in water bath for 57C for 10 mins.
The Rneasy Lipid Tissue Mini Kit protocol is as follows:
  1. homogenize/sonicate samples in 1 ml Trizol
  2. spin at 4C on 12000g for 5 mins and remove fat layer on top
  3. incubate for 5 mins > add 200ul chloroform > incubate 2 mins
  4. spin to separate phases > transfer the aqueous layer to new tube
  5. Add a volume of ethanol to the sample, shake tube for 10s
  6. add sample to spin column
  7. wash with RW1 buffer (to remove carbohydrates, proteins, fatty acids)
  8. add DNase I to digest DNA
  9. wash again with RW1 buffer
  10. wash twice with RPE buffer ( to remove trace salts)
  11. elute in 30ul of 45C RNase free water
My 260/280 is around 1.7 and 260/230 is around 1.
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Our current work uses arthritis model on mice paws and we are searching for the best way to harvest and macerate the whole limb. Maintenance of virus viability and genetic material integrity are the main goals.
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Vicente Mendoza Reyes thanks for your help!
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What's the DEPC water protocol for removing contamination from racks and other utensils in lab?
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