Science method

RNA Isolation - Science method

A forum to discuss and share methods regarding the extraction and purification of RNA.
Questions related to RNA Isolation
  • asked a question related to RNA Isolation
Question
1 answer
A protocol for rna isolation from corona virus
Relevant answer
Answer
This will depend entirely on what your actual sample type is. Oropharyngeal swabs? Tissue culture media? Virus concentrate? Wastewater? Your best bet is probably to look up some papers that isolated the coronavirus RNA from the same sample type you're working with, and use their protocol or something comparable.
  • asked a question related to RNA Isolation
Question
2 answers
Revered researchers , I intend to isolate RNA from oral squamous cell carcinoma surgically resected sample. What is the best and economic kit I can use for RNA isolation.
I found the following:
RNAzol® RT - 50ml = 9.6 INR
GenElute Total mammalian RNA purification kit (70purifications)= 40k INR
RNA isolation kit From Sigma Aldrich = 40k INR.
Please suggest me the one I should purchase. I’m a novice in this.
Relevant answer
Answer
Thank you for your reply Dr Malcolm Nobre
  • asked a question related to RNA Isolation
Question
2 answers
Hi there,
I'm fractionating cells and pulling down a viral protein that binds to a specific RNA. I've been trying to extract the RNA but I have no luck. I have tried basic trizol extraction and direct-zol RNA mini prep with no luck. I think the issue comes when I turbo DNAse treat my sample after RNA extraction. I even clean up the sample and there is no RT-PCR product in my control. What should I do? I was thinking of phase separating the trizol with chloroform first and then adding the ethanol and incubating overnight in -20C before doing the zymo kit. What do you think?
Thank you!
Relevant answer
Answer
Hi Rebecca, recently I met a similar problem. Also direct-zol extraction from RIP without qPCR signal. Although it has passed a long time, may I know whether you have finally found the actual reason? Thanks a lot!
  • asked a question related to RNA Isolation
Question
10 answers
I would like to know if you are using RNase to digest RNA outside EVs? If yes, how do you stop RNase before exosomes lysis to protect RNA inside?
Relevant answer
Answer
Hello, any ideas on how to inhibit RNase A besides using RNase inhibitor?
I read that EDTA does not work against RNase A, nor does heating as it is very stable. Flash-freezing definitely works? Please let me know how to stop it prior to RNA extraction from EVs.
Thank you.
  • asked a question related to RNA Isolation
Question
2 answers
I accidently added five fold more chloroform in the initial steps of RNA isolation. What are the consequences of this? Also, if i add more isopropanol to compensate this effect, how both chloroform and isopropanol are related ?
Relevant answer
Answer
I recommend to start new RNA extraction and get rid of the previous. Because if you are going to continue rescuing your extraction I assume you will face a complicated issues with no result guarantee!!
  • asked a question related to RNA Isolation
Question
6 answers
Hi,
I usually isolate both DNA and RNA with Qiagen AllPrep kits without problem when frozen samples in RLT buffer.
However, I have a sample frozen in RLT buffer without previous removal of cell culture media. Is there any option to rescue the sample?
Qiagen's protocol said: "Incomplete removal of the supernatant will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the Rneasy silica-gel membrane. Both effects may reduce RNA yield."
I have tried to wash the sample with PBS after thawed, then resuspend in RLT and continue with the protocol, but it didn't work.. How can I isolate DNA of this sample?
Thank you!
Relevant answer
Answer
A chance of DNA methylation beside the effect of some hormones or sugars in the medium.Regards
  • asked a question related to RNA Isolation
Question
3 answers
Dear all,
Which is the best method to isolate tumour organoids from methacrylated collagen hydrogel.
I need to isolate RNA from hydrogel cultured tumour organoids.
Thank you for your help!
Relevant answer
Answer
That's awesome! Glad to hear it worked :)
  • asked a question related to RNA Isolation
Question
5 answers
The agarose gel run of RNA samples before DNase treatment gives proper bands for RNA but after DNase treatment, the RNA samples on the gel run band are coming below the ladder size ( I used a 1Kb ladder). I treated RNA samples with DNase at different concentrations of 10 units, 20 units, 8 units of enzyme, and at different incubation time also 30 min. and 25 min. at 37 degrees followed by RNA isolation by Trizol. Yet I am getting the degraded RNA. I am open for your valuable suggestions.
Relevant answer
Answer
Thank you so much Chang and Laura for responding. After RNA isolation I did quantification using Nanodrop as well as ran 0.8% agarose gel to check the quality of RNA. When I matched DNase treated sample and without treated samples of RNA in 0.8% gel the bands were very different. So there is no problem in the protocol but something is definately going wrong with DNase treatment.
  • asked a question related to RNA Isolation
Question
9 answers
I've been working on improving RNA quality lately since I've had a number of bad qPCR results in the past few months. We use the Trizol method in our lab, and recently there has been a lot of precipitation during the RNA isolation. Brief rundown, we homogenize our tissues (I'm using mouse hearts and fats) in Trizol per manufacturer's instructions, followed by a spin to remove insoluble material, and then an addition of chloroform to isolate the RNA. After spinning and removing the aqueous phase, we add it to isopropanol. At this stage, my samples turn extremely cloudy, obviously containing some kind of precipitation. This precipitate can be spun down, and initially forms a gel-like pellet which turns opaque white after washing with 70% EtOH. There is not supposed to be cloudiness at this stage though...the solution is supposed to turn clear after adding the aqueous phase.
I tried this with a blank set of samples (i.e. Trizol without homogenized tissue) and I got the same result...the aqueous phase I pulled off of the blank becomes cloudy after adding to iso, and can be spun down to produce a pellet that looks just like what came out of my tissue samples. I should also note that in my tissue samples, I could see a tiny blue dot after the first EtOH wash (we use glyco blue to precipitate with RNA), so there was definitely a good RNA pellet in there, but it was surrounded by a big flaky piece of white precipitate. The A260/230 ratio is also very low with RNA I get from these cloudy samples, usually around 0.1 (260/280 is good at around 2.0).
Has anyone else had this issue before? Is this some kind of salt precipitation or something else, and how should I get rid of it? I'm guessing maybe the Trizol got contaminated since it happened with blank samples (and I used fresh chloroform and iso, and adding chloroform straight to iso without Trizol did not form this cloudy precipitation), but before I throw the bottle out I'd like to see if this is a known issue I can fix. Any advice is appreciated!
Relevant answer
Answer
That white precipitation is caused By a lot of initial sample used for isolation. I suggest decrease the amount of tissue used for isolation. That will drastically reduce the white precipitate and eventually when one washes with ethanol it dissolves so all gets fine!
Try if this works! It worked for me! thanks !
  • asked a question related to RNA Isolation
Question
4 answers
It's about MagMAX™ mirVana™ Total RNA Isolation Kit. Due to lack of samples, I want to use tubes instead of plates for manual extraction. Protocol suggests, a plate shaker at 700, 950rpm etc etc. Is there any alternative equipment I can use?
Thank you!
Relevant answer
  • asked a question related to RNA Isolation
Question
3 answers
The 260/280 ratio is around 1.8-1.9.
Relevant answer
Answer
Run the ~500ng-1ug of your RNA in either 1-5 or 2% agarose gel in TAE buffer or MOPS buffer RNA gel and check the integrity status. If you find two bands, then you can go ahead.
  • asked a question related to RNA Isolation
Question
6 answers
One of the people in my lab added too much isopropanol to an RNA sample that was isolated with chloroform. What can I add to reduce the percent isopropanol? What is safe to add to an RNA sample/precipitation reaction? The sample was isolated with chloroform, so can I add chloroform to reduce the concentration of isopropanol?
Relevant answer
Answer
Greetings İlknur Albayrak it's because the main purpouse for this is to fractionate the main components in solution, based on solubility. To the best of my knowledge and practice, as the protein precipitation methanol/chloroform, chloroform aims for proteins, while the main objetive of isopropanol is to dehydrate Nucleic acids.
  • asked a question related to RNA Isolation
Question
4 answers
Our lab has some leftover TRI solution and column-based kit for RNA isolation. Is it possible to use them together? Could i use the TRI solution to homogenize the sample then followed by column purification from the column-based kit?
We work with plant samples with some limited access to refrigerated centrifuge. With reduced centrifugation time i could use the regular centrifuge instead.
Relevant answer
Answer
You can try only one sample, as some components of a kit could interfere with other components of the other kit. Best of luck!
  • asked a question related to RNA Isolation
Question
1 answer
As per the kit protocol for the longer durability use RNase-free H2O (with 0.1 mM EDTA) or TE buffer (10 mM Tris, 1mM EDTA) to preserve the RNA for a longer time, but I have a doubt about how to restore the RNA from the above soln?
Kindly suggest the standardized or best protocol.
Relevant answer
Answer
Hi Deepdarshan.
If by restore, you mean you want to precipitate the RNA so that you can resuspend in a different solution at a different concentration, then just use isoproponal or ethanol. Add ethanol at 3x volume or isopropanol tat 2x volume. Then leave overnight at -20C to precipitate the RNA. Then spin to pellet the RNA.
For example. If you take 100 ul of your RNA in the TE buffer, add 300 ul of ethanol or 200 ul of isopropanol. Provided the isopropanol or ethanol is added in excess then it will precipitate the RNA. It will precipitate quite rapidly, but I like to leave overnight at -20C.
best wishes
Simon
  • asked a question related to RNA Isolation
Question
7 answers
Need to store cumulus cells for RNA isolation
Relevant answer
Answer
If you are worried about RNA, take the cumulus without dissociating. Pick up with fine pasteur pipette or pipettor 20-40ul tip (better) Give a rinse in RNALater in a small dish. And pool all the cumuli in 1ml of RNALater in a sterile tube. Follow instructions for storage on RNAlater sheet (enclosed). I will recommend not to dissociate cumulus and not to wash. More manipulations will change your gene expression. After thawing, spin , get rid of RNALater and go with Trizol directly and follow RNA extraction method or any other method you are trying to follow. I have used RNAlater many times, that is very useful.
Good luck
  • asked a question related to RNA Isolation
Question
1 answer
We are digesting mouse cortices and trying to extract the RNA from microglia for sequencing. We have gotten the isolation working to get good yield of cells (about 200,000 for both cortices) but the RNA after flow sorting is having really low RNA integrity scores. We are thinking that this is because of the sort process and are thinking about using RNALater for preservation of cells prior to sorting (i.e sorting with RNAlater as the buffer!). Has anyone tried this? If so - does the RNALater degrade the fluorophore signals? Any other tips for isolating RNA from sorted primary cells would be appreciated!
Relevant answer
Answer
How bad is the RIN? This might be one of the experiments where you have to accept RNA degradation and plan your downstream experiments to compensate for it.
The way RNAlater works is that it actually precipitates the RNA inside the cell. It also denatures a lot of proteins. Typically samples get transferred directly from RNAlater to lysis reagent because if you were to (for example) spin the cells out and put them back in an aqueous buffer, the RNA redissolves, the RNases are active again, and nothing was achieved. I don't think it's possible to put RNAlater through a flow cytometer either because of the salt content.
Some people in my lab did have some success a few years ago - I think they got the RIN up to around 6. If I remember correctly they did it by using obscene amounts of recombinant RNase inhibitor. It might be prohibitively expensive to do that using commercially produced inhibitor, but if you cloned an RNase inhibitor into a plasmid and expressed and purified it yourself, it might be doable.
  • asked a question related to RNA Isolation
Question
8 answers
Hi researchers,
I was wondering if anyone knows a way I could Isolate RNA and Protein from the same cell pellet. I'm not looking for a kit that allows me to do both at the same time unless that is my only option. Basically I have cell pellets I have stored at -80 degrees that range from 800,000-9x10^6 cells per pellet. Because I really only need about 100,000 cells for RNA isolation for qPCR I dont want to waste an entire pellet of cells on just RNA. I need several million cells to achieve enough protein for my western blot analysis. So is there a way I could resuspend this pellet and potentially use like 20% of the lysate/resuspension for RNA and the other 80% for western blot analysis at a later time without damaging the cells or compromising one procedure for another? My big concern is the lysing of the pellet with one type of buffer/breaking the cells and it compromise either of the procedures...
Thanks!
Relevant answer
Answer
Hi Haley,
Probably the trizol reagent is the best option for your problem. You can separate RNA and protein without divide pellet or damage cells.
Here, you can find a protocol of extraction with trizol reagent.
  • asked a question related to RNA Isolation
Question
3 answers
I am trying to isolate RNA from monocytes, but using the Macherey-Nagel Kit (NucleoSpin RNA Plus) I only seem to get concentrations of 10-20 ng/µL RNA in 20 µL elution volume, which is barely sufficient for my follow-up experiments.
I culture 750,000 monocytes in 500 µL RPMI 1640 medium, supplemented with GlutaMAX, NaP, gentamicin and human serum for 24 hours at 37 oC. Then I harvest them by adding LBP lysis buffer to the cells (and resuspending thoroughly) -> freezing them in liquid nitrogen and then storing at -80 oC until subjected to RNA isolation.
How can I get a higher RNA concentration?
Relevant answer
I perform RNA extraction from cells using trizol.
I have used kits with columns, but the amount of RNA is always very low, I recommend using Trizol. Add trizol directly to the culture dish once the medium has been removed. And leave precipitating with isopropanol overnight at -20°C.
  • asked a question related to RNA Isolation
Question
6 answers
I have been trying to isolate total RNA from cattle whole saliva samples for gene expression studies. Saliva samples were collected early morning before feeding to the animal and collected in Trizol for Trizol isolation method and in RNA later for kit method. I have tried both the Trizol isolation method as well as the kit method (Qiagen RNeasy Mini Kit) to isolate RNA from the saliva sample (Pellet as well as Cell-free Saliva) by applying all precautionary measures. After isolation when I run RNA on agarose gel (1%)18s and 28s bands are not visible but I'm getting a band ~100bp. Nanodrop reading OD (260/280) I'm getting around 1.7 to 1.9 with concentration varying from 110-200 ng/microliter. After cDNA preparation, I'm not getting any amplification for GAPDH and ACTB genes. Can anyone suggest where I'm making mistake and how I could enhance my RNA yield?
In the attached gel image Last 3 lanes consist of isolated RNA and the other lanes consist of PCR amplification result of GAPDH and ACTB in which no amplification was observed.
Consistently I'm getting this result for RNA Isolation. Please help.
Relevant answer
Answer
Hello!
RNA rapidly degrades in saliva, so probably you may add some RNAse inhibitors immediately after collecting saliva.
Also you may try adding (spike-in) pure non-degraded RNA of known concentration (e.g. total RNA from other source) so that you may track it through all the process of RNA preparation.
Hope this helps.
  • asked a question related to RNA Isolation
Question
5 answers
my aim was to measure miRNA expression in exosomes. I used TRIzol method for RNA isolation and miRC_LNA_miRNA RT and PCR kit for reverse transcription and qPCR as follows.. however, my qPCR was not successful. most of the tested samples including positive control (U6) were giving me either weird Cq values like 7 to 14ish or not determined at all. Does it mean I did not have enough cDNA generated or what else can be considered to look at?
has anyone had this experience or any recommendations would be great. thanks!
Relevant answer
Answer
Hi
In my case (It was very long time ago); I tried to use miRNA from Qiagen with Syber from Roche and it did not work. Therefore we had to switched to Qiagen Syber for the detection of miRNA expression (which belong Qiagen as well).
I hope this helps.
Best
  • asked a question related to RNA Isolation
Question
2 answers
Hello,
I have extracted DNA from my soil samples (dry and sandy) through two different extraction kit (both from Qiagen)
1) by DNeasy PowerSoil Kit
2) by Total RNA Isolation kit (DNA eluting after RNA isolation through eluting buffer).
I got huge difference in extracted DNA concentration. Total RNA Isolation kit did not have good amount of DNA, but same soil sample had really good DNA concentration when extracted by PowerSoil kit.
Does anyone know what is problem here and which result can be more reliable?
Thanks
Milan
Relevant answer
Answer
Hello Christina,
There is no DNases step in the total RNA isolation kit protocol. In the total RNA isolation kit, just after RNA gets released from the capture column you add another buffer that allows DNA to release from the column. That makes this protocol RNA-DNA parallel extraction. I've been using the same protocol (as per company guidance) to isolate RNA and DNA parallelly from different types of soil samples for a long time. But I never had such a problem.
Even amplicon sequencing was not successful for DNA isolated by total RNA kit but successful with PowerSoil kit.
I measure DNA concentration by a Quantus machine.
thanks
  • asked a question related to RNA Isolation
Question
6 answers
Hi all,
During my qPCR, I run no-RT (NRT) samples and no-Template Control (NTC) samples along with my normal cDNA samples containing animal tissue. However, I got amplification values for all my NRT samples, but none for my NTC control samples, which probably indicates genomic DNA contamination in my RNA samples.
However, the Macherey-Nagel kit which I'm using for RNA isolation contains gDNA removal columns which should normally remove any genomic contamination. Assuming the handling of my samples went fine, what else could have contributed to those problematic results?
I would also be interested to hear any ideas on how to remove gDNA from the rest of my RNA samples - after using the kit and gDNA removal columns - with little to no risk of RNA degradation.
Thank you in advance!
Relevant answer
Answer
There are multiple approaches that you can use. Personally, when I work with RNA and RT-qPCR, I choose primers and/or probes that span exon-exon junctions. When properly designed, this can eliminate the possibility of gDNA amplification signal. Another method that I have seen in literature is to treat your samples with DNAse after purification. You can check your current primer pair(s) with Primer-BLAST to see if they amplify gDNA in addition to RNA. You will need to do two searches. On the input page, under Primer Parameters, enter your forward and reverse primers. Lower on the page, under Primer Pair Specificity, perform your first search with the Refseq mRNA database for your species. Do a second search where you choose “Refseq representative genomes” and pick your species. Compare the results from both searches. Ideally, you will get clear results in the first search (mRNA) and no good matches in the second (genomic) search. Potential amplicons above ~1000bp are less likely to amplify, especially if you shorten your extension time appropriately.
  • asked a question related to RNA Isolation
Question
3 answers
I am trying to isolate RNA from freshly isolated neutrophils from adult blood. Further I maintain culture for 3 hours hours according to my need. I use trizol choloroform method. I got very poor yield, A260/280 is also less than 1.8 even. No result was obtained on agarose gel. Can anyone please share your protocol or suggest me a good idea what will i do. Thanks in advance
Relevant answer
Answer
RNA extraction from sperm-challenged PMNs and control groups
The PMN cells exhibiting NETosis were collected and re-suspended in 100µl of PBS in 1.5 ml MCTs. The cells were mixed with 900 µl of Trizol reagent (Invitrogen) as per manufacturer’s instructions. The RNA pellet was dried in the air followed by dissolved in 30 µl of DEPC treated water and placed in a heating block at 55ºC for 5 min. Extracted RNA was quantified using a NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). The quality and integrity of extracted RNA were assessed by running 200ng of RNA (heated at 65°C for 1 minute) in non-denaturing TAE buffered 1.2% agarose.
  • asked a question related to RNA Isolation
Question
4 answers
Hello researchers. I am using CTAB method to extract total genomic RNA from Citrus leaves. I often observe that the pellet is very minute and keeps floating which makes it difficult to discard the supernatant. I want to know what could be the possible reasons for getting a floating pellet and how to avoid it. Thank you.
Relevant answer
Answer
After adding ethanol/isopropanol (ice-cold) the tubes (2.0 ml vials, I guess) may be spinned at a speed of 12000 rpm or more for 10 min, which should give an intact pellet of RNA (or DNA in case of DNA isolation). Do not use pipette for discarding the supernatant. Simply discard by inverting the tube without disturbing the pellet. Immediately place the mouth of the tube on a tissue paper.
  • asked a question related to RNA Isolation
Question
5 answers
After adding .5 μL isopropanol in my sample i see 2 blurry clouds (one in the bottom and the other one in the top) in my sample. Anyone knows why this happens?
Relevant answer
Answer
Dear Pia, thank you for sharing this interesting technical question with the RG community. Unfortunately I'm not a specialist in this field of research enough to give you a qualified answer because we are inorganic chemists. However, I know that the solutions to many technical problems can be found right here on RG. In this case, please have a look at the answers given to the following closely related question which has been asked earlier on RG:
Cloudiness after adding isopropanol in RNA isolation using TRI-reagent. Why does it happen?
(6 answers)
As you will see, other researchers already experienced the same problems. There is also the possibility to directly contact some of the RG members who provided answers to the 2017 question and ask them for some personal advice.
I hope this helps. Good luck with your work and best wishes, Frank edelmann
  • asked a question related to RNA Isolation
Question
4 answers
I would like to know what the reason of the shape of this curve in the spectrometer after total RNA isolation using Trizol?
Relevant answer
Answer
Please sir, could you add any pictures?
  • asked a question related to RNA Isolation
Question
2 answers
I would like to know what the reason of the shape of this curve in the spectrometer after total RNA isolation using Trizol.?
Relevant answer
Answer
It looks as if the photometer changes the path length at an OD of 15. This should actually be corrected, as the OD should always be given for the same path length. Maybe this is a bug in the software.
Is it reproducible?
  • asked a question related to RNA Isolation
Question
3 answers
Hi,
So the whole blood samples are in RNA later solution and kept at -20 (500 ul blood+ 1000 ul RNA later). Please suggest protocol to discard the RNA later and process for RNA isolation. These RNA samples will be used for both sequencing as well as for cDNA synthesis
N.B: RNA later solution is from invirogen and the RNA isolation kit I will be using is from Qiagen.
Any help would be appreciated
Relevant answer
Answer
Most of the RNA stabilizing solutions are compatible with most of the downstream steps and does not require any additional step to be discarded. As you will probably be using column based extraction there are several washing steps which will remove any traces.
  • asked a question related to RNA Isolation
Question
6 answers
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.
Relevant answer
Answer
I am experiencing the same problem. I think it is not contamination but something with the oligo design.
  • asked a question related to RNA Isolation
Question
3 answers
Which of the two kits is better for RNA isolation from plant rich in secondary metabolites? Maybe you could recommend me something else? I would like to obtain high quality and quantity RNA for qRT-PCR. My material are leaves, stems and roots of Salvia miltiorrhiza.
Relevant answer
Answer
I tried boyh of the kits, and only with NucleoSpin RNA Plant and Fungi Macherey-Nagel I obtained RNA.
  • asked a question related to RNA Isolation
Question
2 answers
Hi, I have been doing RNA isolation using the standard RNAeasy Plus mini kit. The 260/280 ratios seem fine - between 1.8 - 2-0, however the 260/230 ratios have been consistently low ( below 1.. as low as 0.3) or extremely high( above 2 or even 3) and I am unable to figure out why. I was using the same kit in my previous lab and following the protocol but I never had this issue. I do the extra spin step with an empty eppendorf to make sure that I have dried the column before elution with water.. I tried running a few of the samples on the Bioanalyzer to check the RIN values but the samples look fine..
I asked around in the department but either people were not facing this problem or they told me it did not matter so much .. so I am confused.. AND curious .. as to why this keeps happening with my samples.. what could i be doing wrong.. ? when I measure the RNA amounts on the nanodrop, I always keep the samples on ice and then I store them at -80deg C.
Would appreciate your suggestions / thoughts.
Thanks,
Zankruti
Relevant answer
Answer
I am currently facing the same problem with the Qiagen miRNeasy kit. Although some samples have adequate 260/230 ratios, most have very low purity and it seems to be just random. Nanodrop One shows that the problem is with Guanadine salts contamination, which are components of the wash buffers. I tried to dilute the buffers with absolute ethanol by double the instructed dilution and add an ethanol wash step before dry spin. Initially some samples had better purity but then others didn’t fare as well and again it seems very arbitrary. The only thing that can reasonably be to blame for this random poor purification is bad non-standardised spin columns that cause droplets of the wash buffers to be caught inside the column however long you centrifuge your samples and contaminate the RNA upon elution. I would opt for extraction by TRizol solely but it has its own shortcomings, too.
  • asked a question related to RNA Isolation
Question
13 answers
Hi, I want to ask that QIAGEN ask following steps to do but if we dont have needle & syringe so can we directly put RNase free water into DNase I stock ?? If not so what is alternative of this? ''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.
My 2nd question is that in this step we keep it at Room temperature for 15 min? or centrifuge it at 20-30 degree for 15 min?
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.
Relevant answer
Answer
Hello Bushra Khattak could you please tell me what is the solution you found for the RNase-free needle and syringe? I have the same problem now.
  • asked a question related to RNA Isolation
Question
2 answers
Is its advisable to perform RNA isolation (By Trizol or Using kits) after staining the animal tissue culture cells with live / dead staining kits?
Relevant answer
Answer
Thanks Sir for your kind suggestion.
  • asked a question related to RNA Isolation
Question
3 answers
Today I was isolating RNA from human atherosclerotic plaque tissue. During the process, I found the interphase extremely thick. Does anyone know of a reason what could cause this? Does it have further effects on the RNA isolation?
Relevant answer
Answer
The interphase of a Trizol-based RNA extraction typically captures the DNA while proteins are trapped in the lower organic layer. The interphase thickness will generally correlate to the amount of cells/tissue that are used. You should do your best to avoid collecting any interphase or organic phase as these will reduce the purity of RNA due to DNA or phenol contamination. However, a substantial interphase is good to have as it will minimize the chance of phenol contamination.
Since you are doing a high lipid content tissue, it may also be beneficial to remove any visible fat layer present at the top of the phenol after tissue homogenization. (see )
  • asked a question related to RNA Isolation
Question
5 answers
Hello everybody,
I am wondering if it is possible to isolate RNA from freeze-dried human tissue in high qualitiy in such a way that RNA sequencing can be done from the samples. Does anybody have experience with that?
Thanks a lot!
Relevant answer
Answer
Yes, Zaidah, both are the same thing, samples have to be frozen all the time during drying and vaccume process
  • asked a question related to RNA Isolation
Question
1 answer
Hi everyone, I am dealing with C2C12 lineage, and I am standardizing the number of wells needed for PCR analyses. I tried to isolate the RNA with trizol protocol for cells, although until now I didn't find any articles that explicit show the exact RNA yield I will need to found at the last step of quantification. I only found it for fibroblasts, epithelial cells. I wish someone can help me about it.
Best wishes for everyone.
Relevant answer
Answer
This paper indicates total mRNA yield per number /size of cultures. Is this what you are asking?
“Seed 150,000 cells/well in a 12 well plate” “Culture C2C12 cells in complete DMEM until they reach ~95% confluence.”
“A typical yield from 1 well of a 12 well plate is 5–7 µg of total RNA with an A260/A280 ratio of 1.8–2.”
  • asked a question related to RNA Isolation
Question
3 answers
Hello Everyone,
I am working on a 3D hydrogel system. I plan to study changes in gene expression in my 3D hydrogel sample. My question is, can I isolate cells from the hydrogel, then culture for 6hrs and then initiate my RNA isolation and downstream assays? What are other possible methods to isolate good quality RNA?
Thanks in Advance!
Relevant answer
Answer
Retrieving cells from the hydrogel system will depend on the hydrogel's nature and composition. RNA isolation can be possible by dissolving the hydrogel and take the cells for RNA isolation directly.
Retrieving the cells from the hydrogel and then culturing and then RNA may complicate the process. Based on the hydrogel composition, surely there will be another agent which will dissolve the hydrogel and you can go ahead with RNA. So, you can find out that solution.
We have done this with Sodium alginate encapsulated beads, we decapsulated the beads using the decapsulating solution and collected the cells for the experiment. Well, we performed cell viability assays therefore we were sure that cells were alive within the beads.
Regards
Saurabh
  • asked a question related to RNA Isolation
Question
3 answers
I have been using the Qiagen's RNeasy kit for RNA isolation from mealybug "Maconellicoccus hirsutus". Though the 260/280 ratio always lies between 1.8-2.0, the 260/230 values are always less than 1 and sometimes even between 0.2-0.5.
Is it because of wax coating on the adult females?
Relevant answer
Answer
High absorbance at 230 is usually guanidium salts (most RNA extraction methods use guanidium isothiocyanate). These are chaotropic, so not good for proteins: too much salt makes your reverse transcriptase work less efficiently (or not at all), consequently affecting all your downstream analysis.
If this is the case here, you can usually remove them by simply reprecipitating your RNA (especially when you have a good RNA yield, as here). You'll lose some RNA, but will lose almost all salt.
------------------------
Make your sample up to 500ul with DEPC H2O (optional, but it's a volume based protocol, so using larger volumes makes everything easier).
Then add 50ul (1/10 vol) of 3M sodium acetate, pH 5.5 (autoclaved), and ideally 10ug of glycogen (to improve recovery). Mix.
Then add 500ul of room temp isopropanol (1 vol). Must be room temp, not chilled (cold isoprop will precipitate salt). Mix well, incubate at room temp for 20mins.
Spin at max speed in a chilled centrifuge, 15mins. Tube hinges outward, so you can see where the pellet should be (in case it's small)
Remove supernatant carefully, add 300ul of ice-cold 70-75% ethanol.
Spin again, 10mins.
Remove supernatant, repeat with another 300ul of ice-cold 70-75% ethanol, spin (10mins), remove the supernatant completely, leave in a fume hood to dry for ~10-15 mins.
Redissolve in an appropriate amount of DEPC H2O (I usually assume recovery will be about 70% of my starting material, so adjust vols accordingly).
  • asked a question related to RNA Isolation
Question
1 answer
Hi!
I'm trying to improve some protocols for RNA isolation from (among others) adipose tissue . I'm trying to figure out what are the best binding conditions for RNA using qiagen silica columns.
I did the extraction of RNA using combined protocol from different publications and qiagen kits which use trizol(qiazol etc) + chloroform + columns. Results were not good, especially the 260/230 proportion is very low eg. 0,3 so it cannot be used in qPCRs.
Also I tried two step method for RNA isolation - first homogenisation with buffer cont. Gu-HCl, Tris-HCl and triton x-100 pH 6,6 then after centrifugation supernatant was applied to qiagen column to theoretically bind DNA (?), then EtOH was added to the flow-through to precipitate RNA (is it better to precipitate it through the night at -20?) and washed with buffers from Roche or Qiagen. Of couse I know that they might appear DNA contamination but it will be removed with dnase treatment. RIN are very good - 8,2 or even higher usually nothing less than 7, but the concentration is very low (20ng/ul) and 260/230 too.
So I'm looking for some advices, books, articles, websites - everything :) to improve my work. Or maybe someone have any ideas or can explain me some things?
What's yours expierience with trizol-chloroform-columns extraction? Did you do the two-step RNA isolation on columns?
Relevant answer
Answer
Hello,
I think using the following protocols will bring you good results.
I used the Guanidine/Phenol solution for total RNA isolation and got very good results.
  • asked a question related to RNA Isolation
Question
4 answers
I am doing RNA isolation using QIAzol lysis reagent. After collection of upper aqueous phase, it says add 1.5 volumes of 100% ethanol? how much do I need to add and how to prepare?
Relevant answer
Answer
'Add 1.5 volumes' means that whatever the volume of your collected aqueous phase is, you add 1.5 times that much. So if your aqueous phase is 400uL, you would add 600uL of 100% ethanol.
  • asked a question related to RNA Isolation
Question
6 answers
I have about 100 blood samples in edta tubes, I keep these tubes at -80 degrees. I'm going to do gene expression from them,
Has anyone used GeneJET Whole Blood RNAPurification Mini Kit
#K0761 and had good results? Or should I do RNA isolation with trizol?
Relevant answer
Answer
Kaled as i understand you are getting serum not whole blood,
The blood samples I will use are stored at -80 degrees for about 2 months.
I hope I get results in rtpcr,
Best wishes
  • asked a question related to RNA Isolation
Question
6 answers
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.. 
Relevant answer
Answer
Any kit suggestion for smallRNA from single cells?
  • asked a question related to RNA Isolation
Question
3 answers
Can I store mouse ear tissue homogenate at -80 before RNA isolation using Gene JET RNa purification kit(cat no# K0731)?
Relevant answer
Answer
It‘s ok. But you'd better homogenize the fresh tissue immediately after collecting it and avoid repeated freezing and thawing.
  • asked a question related to RNA Isolation
Question
5 answers
Using H9 hESCs I have developed neural progenitor cells using SMAD signalling inhibitors, then differentiated them into neurons by excluding EGF and FGF from the media and plating onto laminin coated plates. I have very nice neural networks in the dishes but when I go to isolate RNA using Qiagen RNA easy mini kit, I dont get any RNA. I was wondering if anyone else has had this problem with RNA isolation from neurons and if there's anything I can do to rectify? Thanks in advance for any advice!
Relevant answer
Answer
I have an additional question here. Is it okay to freeze these types of cells down at -80 for a future RNA extraction?
  • asked a question related to RNA Isolation
Question
4 answers
I'm planning to isolate RNA from cells stored in RNAlater with Qiagen mini kit. In one article, I saw we can directly use cells in RNAlater without washing it with PBS (but needs to put a higher volume of lysis buffer). Does anyone has done that?
Relevant answer
Answer
Dear Nirmani, if your cells are stored in RNALater at - 80 C then after thawing suspend them at 2500 g at 4C for 5 minutes then discard supernatant to get rid of RNALater solution and start extraction by adding up the lysis buffer, whereas, if you had stored your cells in the RNALater at -20 C then after thawing suspend them for 10 minutes at 5000 g at 22 C, then discard supernatant and start your RNA extraction. Volume of cells to be stored in RNALater is 1 * 10^5 - 10^6 / ml in 200 ul RNALater, but it is always advisable to get your pellets washed with cold PBS first then freeze them at -80c or -20C by adding up the RNALater. It is the recommended protocol for cell pellets. Hope it helps....
Regards....
  • asked a question related to RNA Isolation
Question
5 answers
We have a RNA collection in our freezer and wondering if it’s worthwhile keeping this study or throwing it away to create space for other studies.
The RNA was isolated 20 years ago from heparin blood tubes. The time until RNA isolation differs between samples from 2 to 5 hours. The RNA has been stored at -80C.
We know it’s best to use Paxgene tubes to stabilize the RNA. Would any of you still use these RNA samples for transcriptome studies or any other types of studies?
Does the heparin have an influence on the outcome in any way?
Many thanks in advance.
Relevant answer
Answer
Tools exist specifically to check RNA quality: measuring the RIN of a selected few samples (bioanalyser/tapestation) would tell you whether the RNA is still intact and of sufficient quality and quantity.
This is far, far cheaper than taking it forward to transcriptomics without checking.
  • asked a question related to RNA Isolation
Question
3 answers
Hi,
I want to delve deeper into nanopore sequencing and bacterial transcriptome analysis and as I don't have that much experience in this area I am basically venturing blindly.
I isolated my RNA from E. cloacae using column Zymo kit. Then I tried to measure my results in the Tapestation, however I obtained suprisingly low RIN scores (RIN: 4-7). I recheck my samples for DNA contamination and RNase with Qubit and my samples are fine. Obviously I have spikes where I should not have them but what would possibly cause my RNA to be so damaged?
Is anybody has any idea what could be the issue? I am attaching my Bioanalyzer results from 2 bad samples and one that is slightly better.
Thanks for any help in advance!
Relevant answer
Answer
Ok, I'm seeing a few things here that might be relevant so I guess I'll go down the list. I'm more familiar with the Bioanalyzer than the Tapestation but they are similar enough that I think most of this should apply to your results.
Firstly, as Aditya has already said, RIN scores are calculated on the strength and relative abundance of the rRNA peaks - 16S and 21S in the case of prokaryotic samples. Large RNA is more prone to degradation than smaller RNA. With intact total RNA, the large subunit (21S) can be almost double the peak height of the small subunit. As the RNA gets progressively more degraded, the ratio equalises then inverts - you can see this in your samples. Because most of the degraded RNA ends up as small fragments, there will also be a peak of fragmented junk at the very small end of the distribution, which will be relatively larger in more degraded samples, and I can see this in your traces too. I don't know what the big spike is in your middle quality sample in between 16S and the junk peak.
Secondly, your samples are overloaded - the height of the junk peak is cut off in all of them. This will affect quantification results and may affect RIN so interpret with caution if you see any peaks cut off.
As for the causes... I am used to working with eukaryotic material. Eukaryotes keep their endogenous RNases partitioned away from their RNA in cellular compartments that prokaryotes don't have. I really don't know how much of a contribution endogenous RNases (that were expressed by the organisms you're working on) make to RNA degradation in prokaryotic samples, versus contamination with environmental RNases (the ones that are hanging out in your lab - and there will be plenty!) You can start by taking general precautions against RNases, namely: Use separate chemicals, buffers, water etc. for RNA work, and handle them with great care. Never touch or open them without gloves, leave them open, use them while bacterial cultures are on your bench etc. Clean your work area, pipettes, centrifuges, etc. with RNase inhibitor spray before you start RNA work. Only use plasticware, chemicals etc. that say they are RNase free.
For your extractions, do your best to minimise the amount of time between cell disruption and RNA extraction. (Make sure you are following a protocol for RNA extraction from bacteria that includes a lysis step, rather than a lysis protocol first and then a RNA extraction protocol.) Keep buffers chilled and work on ice. Don't leave anything to sit around for longer than the protocol specifies. Elute your RNA in ultrapure water and put it immediately on ice.
Hope some of this helps :)
  • asked a question related to RNA Isolation
Question
4 answers
I am doing a dissection to get a small amount of tissue for RNA isolation. The saline was made using non RNase-free/sterile parts. Also, the area I am dissecting is very close to the gut which occasionally becomes permeated so there is a likelihood of high levels of RNases during the dissection.
Somebody suggested I add BME to the saline to inhibit RNases. My question is what would be a sufficient amount to add to inhibit RNases without making it immediately toxic to the tissue I'm dissecting.
Relevant answer
Answer
For maximising RNA quality from fresh tissue, the single most important thing you can do is keep it really cold. Whether you decide to use supplements like BME or not, do your dissections in ice-cold buffer or on a chilled metal platform (I use a stainless steel lunch box packed with ice slurry - I flip it upside down on the bench and use the underside as my dissecting platform.) For my tissue type (mouse brain) we have found that adding RNase inhibitors helps a lot. 1uL/mL of 1M DTT and 1uL/mL of RNaseOUT added to the buffer for dissection/storage, combined with keeping everything ice cold, lets us get high quality RNA (RIN 8+) sometimes after half an hour or more of handling and dissection steps between death of the animal and beginning RNA extraction.
  • asked a question related to RNA Isolation
Question
3 answers
I am looking for synthesizing cDNA from total RNA, isolated from wheat leaf. I have GoScipt Reverse Transcription Kit. After some searching, I have found that this MMLV reverse transcriptase does not have RNaseH activity nor the kit has any separate RNaseH enzyme.
Can I use this kit for synthesizing full length cDNA for cloning purposes? Or I have to use separate kit/ RNaseH?
N.B. My target gene is 1.6Kb in length
Relevant answer
Answer
The RNase H activity of reverse transcriptase acts as an endonuclease that hydrolyzes the RNA strand in an RNA/DNA hybrid to generate 5′ phosphate and 3′ hydroxyl ends. Please see the attached document for detailed description.
  • asked a question related to RNA Isolation
Question
4 answers
Hi, for my experiment I have to seed the cells on the lower (abluminal) side of the inserts (no cells on regular side). I am facing issues with isolating RNA from those cells. Anyone has any experience? There should be around 300,000 cells (on 0.4 um Falcon 12-well format inserts). I have tried Qiagen RNeasy columns for RNA isolation. Thanks.
Relevant answer
Answer
I am just asking out of curiosity!
Did you think of scrapping it from the bottom? Or use TRIzol and lyse the cells from the bottom?
Also, if you are not growing any cells inside the insert, why don't you flush it using TRIzol from inside the insert and collect the cells in a new 12 well culture dish?
  • asked a question related to RNA Isolation
Question
3 answers
Hi I'm isolating RNA from mycobacteria for qPCR
so I added 500ul of Trizol to cells and got rid of cell debris with centrifuge
after that I added 300ul of chloroform and centrifuged 5min at 16000g, 4℃
but i realized that aqueous phase was so cloudy and the interphase and aqueous phase wasn't separated properly
so it was really hard to get aqueous phase without cloudy things
should I centrifuge longer than that?
is there anyone who experienced similar problem?
Relevant answer
Answer
Hi,
I had similar experience. I would advise you centrifuge your mixture using a low/moderate speed(rpm) centrifuge. Then, you will be able to obtain a clearly separated layers.
  • asked a question related to RNA Isolation
Question
3 answers
Hello Everybody,
I wanted to start this discussion to gather the experiment results from whoever have gone through this.
I am trying to extract total RNA from both serum and plasma, continue with cDNA synthesis and qPCR.
I am using grade II or III cancer patients’ blood sample.
I am on my validation period and still working on it.
Could you please share your experiments in this field ?
All you experiments such as:
- Serum and Plasma isolation protocol,
- Amount of serum and plasma used in RNA isolation,
- RNA extraction kit? Did you use specific kit for RNA isolation from serum/plasma or any other commercially available ones?
- Any key point in cDNA synthesis,
- Probe or syber green, which one did you chose for qPCR experiment?
- Any commercially available qPCR master mix or more specified one?
Thank you
Relevant answer
Answer
For extracting cfDNA you should utilize the assay with cfDNA extraction kits or viral DNA extraction.
For RNA assay you should add RIBOX or sth. like it, to prevent RNA from degradation.
qPCR is not an appropriate method .... you should quantitate it with the methods like HRM because of your goal (if you want to study the DNA methylation)
  • asked a question related to RNA Isolation
Question
3 answers
Hi there, I'm looking for help to deposit RNA from DEPC-treated water.
I used Trizol to do the total RNA isolation and finally dissolve the RNA in DEPC-treated water. However, I found I did a wrong calculation about the volume of the DEPC-treated water, which cause the low concentration of RNA. How can I make the RNA be precipitated from the DEPC-treated water? It would be pretty kind if someone can help me out.
Relevant answer
Answer
Katie A Burnette Thanks for your help. I'm a little worried about the RNase contamination and the following RNA degradation if I open the lips. I'm considering whether I can add isopropanol in the DEPC-treated water just like depositing RNA from the aqueous phase after extraction with Trizol and chloroform.
  • asked a question related to RNA Isolation
Question
1 answer
I am trying to extract RNA from Aedes aegytpi to analyze gene expression in genes associated with the detoxification of insecticides. I am using the SV Total RNA Isolation System from Promega, but I got a very low yield of RNA (from 30 - 50 ng/ul). Can you share any tips or protocols or how have you solve low yield in RNA isolation from mosquitoes. Thank you very much.
Relevant answer
Answer
For RNA extraction you can use, Applied Biosystems® Arcturus® PicoPure® RNA isolation kit (Arcturus, biological Biosystems, USA), you can remove the DNA remnants with the Baseline-ZERO ™ DNase removal kit (Epicenter, Illumina) and the ribosomal RNA with the Ribo-Zero ™ rRNA removal kit (Human / Mouse / Rat) (Epicenter, Illumina) these protocols provide high RIN
  • asked a question related to RNA Isolation
Question
9 answers
Dear all,
I am trying to run a qPCR on RNA isolated from FACS samples but it seems that the quality of my RNA / cDNA is not high enough and the qPCRs keep failing. My primers are not the issue since they work on RNA isolated from tissue.
I isolate the RNA thanks to the QIAGEN RNeasy Micro Kit and already tried to improve the quality of the isolation by:
- Combining the RNeasy Micro Kit with a Trizol RNA extraction
- Directly collecting the cells in Trizol to avoid its dilution in buffer
- Extra PBS washing steps before proceeding with the RNA isolation
Unfortunately, this didn't improve the qPCR results.
Would anyone have had the same issue and could find a solution?
Thank you.
Relevant answer
Answer
Thank you! Please let us know if there is any improvement! Good luck!
  • asked a question related to RNA Isolation
Question
5 answers
Hello everyone! One of my students asked me a question about DNAse step during RNA isolation. She asked me how is it possible, that DNAse is responsible for hydrolyzing phosphodiester bonds in DNA, and it does not degrade the same bonds in RNA, although it is a less stable molecule? I haven't found any precise answer, so I would be very grateful for your help!
Best regards,
Joanna
Relevant answer
Answer
Looking at DNase 1 structure bound to DNA, the 2' hydroxyl of RNA is geometrically blocked. In particular a tyrosine packs against the ribose sugar which would directly collide with the 2' hydroxyl of RNA. So this collision should preclude excision of RNA but allow DNA 2' deoxyribose devoid of oxygen in its 2' position. In general geometric or steric collisions provide exquisite specificity in molecular biology by docking non-target interactions.
  • asked a question related to RNA Isolation
Question
2 answers
My problem is on the purity of my RNA. When tested on nanodrop, the A260/280 ratio is around 1.8 - 2.0 which is good; however, the 260/230 ratio is low, around 0.4-0.5. Any tips on how I can purify my RNA? FYI? I'm using Trizol reagent.
Relevant answer
Answer
How about a kit like the RNeasy mini kit?
  • asked a question related to RNA Isolation
Question
4 answers
I guess there should be a washing/equilibrating step (buffer) and an elution step (buffer). I have tried direct elution with warm TE with limited success.
Relevant answer
Answer
you can try to use 95 degree pre warmed TE buffer. It might work better than just a bit warmed buffer. But before it you need to wash it with wash buffer to eliminate many other conteminants. In addition if that method doesnt help you can add 0.3 M NaCl in your elution buffer. that will disassociate DNA from column.
  • asked a question related to RNA Isolation
Question
9 answers
I have used Promega RNA kit, but it didn't work. The volume of CSF that was used was 6ml.
Relevant answer
Answer
Johannes Denk Kindly suggest what will be the better way to isolate RNA from snap-freezed CSF samples
  • asked a question related to RNA Isolation
Question
4 answers
1. We have extracted DNA from nasal swabs and BAL using the Qiagen DNA Blood and Tissue Kit for extraction. There is now a Qiagen QIAamp DNA Microbiome Kit available. Has anyone compared DNA yields with the DNA microbiome kit versus the Blood and Tissue kit?
Relevant answer
Answer
Hi. Hope this is still relevant. Can someone give me some details on the protocol for DNA isolation from NP swabs? How much DNA do you get?
  • asked a question related to RNA Isolation
Question
1 answer
I have been having great difficulty in isolating RNA from bovine preantral follicles despite many changes to protocol. Our goal is to isolate RNA from a single follicle with ~100 cells, but even with 10+ follicles pooled, our RT-qPCR results have been negative for both beta-actin and aromatase expression but is able to detect expression in a single oocyte with a few granulosa cells attached. We believe there is a lysing issue with the follicles due to the basement membrane. I have tried: 2 different single-cell RNA isolation kits, using collagenase/pronase for 30 mins before lysis step in RNA isolation, adding more cDNA, increasing to 40 cycles, low retention tubes, using Cells-to-cDNA kit, and using RNA-to-PCR one step kit. Any advice for getting this to work is greatly appreciated!
Relevant answer
Answer
Trizol method will work. I have tried and also got good RNA concentration from 3 buffalo preantral follicles.
  • asked a question related to RNA Isolation
Question
5 answers
Hello,
I am a Master's student working with Murine Skin samples for qPCR analysis. I am having a hard time getting clean ribosomal bands with my RNA- some appear but are still VERY smeared. I have good A260/280 ratios (around 2). I am homogenizing using a drill and lysis buffer + Proteinase K for 30-45 minutes. I then pull off the supernatant and isolate my RNA.
I have attached an example of what my bands look like. I seem to be getting good amplification from the cDNA made from the RNA, but I would like to clean it up before I publish with it.
Thank you in advance!
Relevant answer
Answer
Hi everyone, I figured out the issue- the skin samples were not being lysed enough. We had been using drill bits with lysis buffer and we had to switch to a bead bashing machine.
  • asked a question related to RNA Isolation
Question
4 answers
For several months, we have been collecting whole blood from patients then using density gradient centrifugation to isolate mononuclear cells (MNCs). We then immediately add Qiagen's RNALater to the cells and store them at -80. We have started trying to isolate genomic DNA and RNA, the first for HPLC and the second for microarray. However, there are red blood cells and (probably) hemoglobin still present that clogs up Qiagen's AllPrep DNA/RNA Mini Kit columns. We tried starting with the first few steps of the Trizol method to help clean the samples prior to launching into the kit, but were wondering if there is a better way. When speaking with a Qiagen rep, my colleague was told that the their red blood cell lysis buffer was not compatible with the RNALater we have been using.
Relevant answer
Answer
following
  • asked a question related to RNA Isolation
Question
3 answers
I will be performing RNA isolation with frozen lung biopsy samples and am in the market for a tissue homogenizer. I am wanting a homogenizer that will fit in a 1.5ml tube. I was looking at the Fisher 150 Homogenizer (15-340-167), as well the 7mm Fisher saw-tooth probe (15-340-174). If anyone has experience using these products or knows of another product that will work, please let me know.
Relevant answer
Answer
I typically do a few rinses in 50 mL tubes between use with brief activation of the probe in each:
  1. ddH20
  2. 15% hydrogen peroxide
  3. ddH2O
After this I disassemble the probe to check/remove remaining tissue. Reassemble the probe, and dip the tip in Trizol/Qiazol (which is what I perform my homogenizations in).
I only use RNAse Zap on the workspace and probes before I begin the first homogenization. You can definitely include that as an extra step during the cleaning between samples if you want, just make sure you follow it with a water rinse.
I've attached my complete protocol if you are interested.
  • asked a question related to RNA Isolation
Question
9 answers
Hi, I have been struggling with a weird issue for my RNA samples. I used the Qiagen RNA plus mini kit. For some reason no matter how careful I am with the washes, when I measure the RNA, I get really low values for the 260/230 ratio, on average around 0.5 or below. I follow all the steps suggested in the kit.. I thought that it must be the Ethanol which causes such ratios so I try to adequately dry the column with an extra spin.. but that does not seem to help.
I checked the quality of my RNA on the Bioanalyzer and it seems the quality is fine. Even the yield of RNA from my samples is decent .. ( I grow cells in 6 well plates ).
Relevant answer
Answer
Carryover of chaotropic guanidium salts is pretty common with kits, in my experience.
If your yield is good, you can usually clean up your RNA by precipitation:
Make up vol to 500ul with nuclease free water
Add 0.1 vols (50ul) of 3M sodium acetate, pH 5.5
Add 10ug of glycogen (optional: increases recovery yield)
Mix.
Add 1 vol (500ul) of room temperature isopropanol.
Incubate at RT for 20mins, spin at max in a chilled benchtop (4 deg) for 15 mins.
Wash 2x with ice cold 75% ethanol (add ~300ul gently to wash over the pellet, spin after each addition).
Air dry for 15mins or so
Redissolve in water.
This approach will generally lose ~20-25% of your RNA, but 90-95% of your salt, giving you much cleaner, usable RNA.
Room temp is important, btw: cold isopropanol will precipitate salt too, which you really don't want.
  • asked a question related to RNA Isolation
Question
3 answers
I have limited amount of samples, so I am trying to get RNA, DNA and protein from the same samples. I used Trizol reagent for RNA isolation, then use back extraction buffer for genomic DNA isolation. After back extraction I saved the viscous pellet, I have seen people save it for protein isolation but was not able to find a protocol to proceed. Can anyone help me with this?
Relevant answer
Answer
There are a few kits that allow you to isolate RNA, DNA and proteins from the same sample. I have tested Quick DNA/RNA mini prep from Zymo after binding the RNA to the column you can precipitate the protein content with cold acetone. Check this protocol:
  • asked a question related to RNA Isolation
Question
8 answers
Currently, I have a problem to isolate total RNA from mouse primary cortical neuron culture. I am seeding neurons to poly-L-lysine coated coverslips and seeding 500k cells per coverslips in a 6-well plate. For RNA isolation, I was scaping 3 coverslips under 1 ml trizol. After that, I was proceeding with Direct-zol RNA isolation kit protocol. But there was no RNA or sometimes very little RNAs such as 20ng/ul after isolations at many times. Cells were looking confluent and healthy before scraping. Therefore, I am not sure about which stage the problem occurs. I would be so grateful to get an extra idea/tip about this. Is there anyone doing RNA isolations from primary neuron culture and managed to solve this kind of problem?
Relevant answer
Answer
Hi Ayca,
I agree with Suraiya that in theory for your purpose it would be better to grow the cells directly in the well plate e.g. as a mixed culture containing glia and neurons. We had problems once with scraping cells from coverslips. However, still I think that with your feeder-approach having the neurons on coverslips you should be able to extract more then enough RNA. I was currently using a mixed culture of 800 k cells per 6 Well. Cells were harvested at DIV9 or DIV21 using QIAGEN lysis buffer and RNA extraction was perfomed with QIAGEN RNeasy Kit. In the past, I made best experiences with the RNeasy kit. I also combined it with TRIZOL lysis and extraction and it worked very well. I don't have experiences with the kit that you have mentioned. However, with the RNeasy kit I usually get 3-5 µg total RNA of high quality (RIN>8) from these 800 k cells.
Maybe you could try this.
Best
  • asked a question related to RNA Isolation
Question
4 answers
Hi all!
My university is starting to apply a new phenol-free policy and I am trying to find a phenol free RNA-isolation kit. It would be for brain tissue. Normally I use Trizol/Qiazol reagents getting a good yield. If anybody is using one that it's working well for brain, please share :)
Many thanks in advance!
Relevant answer
Answer
Thank you very much for your answers!
Just one thing, Junaith S. Mohamed : I was using the RNeasy Mini Kit from Qiagen combine with Trizol (as the first step of the protocol) to disrupt and homogenise the brain tissue. May I ask you what do you normally use for that? Maybe the "RNAprotect stabilized tissues" from Qiagen?
  • asked a question related to RNA Isolation
Question
7 answers
We're doing a RNA phenol/chloroform extraction from individual zebrafish embryos and using a phase lock gel to get the best possible RNA quality. We'd like to also be able to genotype the embryos by DNA extraction after this. I've seen protocols that allow back extraction of DNA after RNA isolation, but they don't use a phase lock gel. Anyone have any idea if this can be done still using the gel? 
Relevant answer
Answer
Jumping onto an old post, but is it possible to reuse the phase lock gel with the organic phase underneath by adding back extraction buffer for DNA precipitation directly into the mixture and then mixing the whole thing again, assuming the mixture will mix through the gel layer? (via puncture or something to allow the gel layer to dehisce?)
  • asked a question related to RNA Isolation
Question
7 answers
Hi Everyone,
I did a few rounds of RNA isolation today and found something very strange. I usually have very good A260/A230 ratios along with A260/A280 ratios, but this time around my A260/A230 ratios were VERY low. I am not sure what went wrong but it was consistent among the two times I isolated today.
Could this be due to the reagents in my RNA isolation kit being contaminated? This doesn't seem likely to me but this is really the only variable that has changed considering I have moved on to use the newer kit now that I am proficient at isolation.
In my second run today I tried eluting two rounds with water at the end which helped a little but the ratio was still far too low. It also dropped my concentration down to about 30 ng/ul from 100 ng/ul.
In past isolations, my concentrations would range from 400-1000 ng/ul, so this was very strange.
Tomorrow I plan on trying it again with a different kit to see if that was the issue.
Any suggestions or comments would be appreciated, thank you!
Relevant answer
An abnormal value (high or low) of 260/230 may indicate a problem with a sample or with an extraction procedure. This info may help
1. A low A 260/A230 ratio may be the result of:
• Carbohydrate carryover (often a problem with plants).
• Residual phenol from nucleic acid extraction.
• Residual guanidine (often used in column-based kits).
• Glycogen used for precipitation.
2. A high A260/A230 ratio may be the result of:
• Making a Blank measurement on a dirty pedestal
• Using an inappropriate solution for the blank measurement. The blank solution should be the same pH and of similar ionic strength as the sample solution.
Example: Using water for the Blank measurement for samples dissolved in TE may result in low 260/230 ratios.
Reference:
William W. Wilfinger, Karol Mackey, and Piotr Chomczynski,
Effect of pH and Ionic Strength on the Spectrophotometric
Assessment of Nucleic Acid Purity: BioTechniques 22:474-481
Merry Christmas
  • asked a question related to RNA Isolation
Question
2 answers
I am trying to determine what best time-point to harvest my cells, HMVEC, after transduction with long non-coding RNA to determine changes in mRNA levels, specifically VEGFR2/KDR.
I am capable of achieving ~90% transduction efficiency by assessing GFP+ cells via fluorescent microscopy as my adenovirus is tagged with GFP. My current protocol is:
Day 0: Seed cells
Day 1: transduce cells with adenovirus (24 hours post seeding)
Day 2: change media (24 hours post transduction)
Day 3: harvest cells
What would be an ideal time point to harvest cells to determine changes in mRNA levels? I was thinking between 16-24 hours post transduction. Would appreciate any input or advice, thanks in advance!
Relevant answer
Answer
Hi Mark,
Since you can achieve ~90% transduction efficiency according to GFP+ cell assessment, I would recommend you to detect mRNA expression of your genes of interest at different time point (12 h, 16 h and 24 h, with triplicates per time point). There you might know at which time point it's optimal.
In our previous studies, we infected human SMC instead of HMVEC with adenovirus (MOI=20), normally after 24 hr of infection we changed fresh medium and treated the cells with vehicles or drugs. 6 h or 12 h after drug treatment we collected cells pellets for RNA isolation and further mRNA expression in response to drug treatment or RNA sequencing.
Hope it works for your experiment.
Best
  • asked a question related to RNA Isolation
Question
6 answers
Hello, 
I am about to IP my protein of interest and maybe (I hope so) some small RNA would co-IP with it. Since this is my first time in this area, would anyone be so kind to suggest the best method for separating these RNAs for the further analysis (based on your own experience [good & bad])? I am thinking of trizol but not really sure.
Thank you in advance!
Relevant answer
Answer
Dooyoung Lee could you please share your IP-RNA extraction protocol?
  • asked a question related to RNA Isolation
Question
5 answers
I extracted total RNA from fungal samples and my goal is to send the RNA for RNAseq, however my RNA did not pass the quality control due to atypical baseline by the bioanalyser results (Please see image attached).
The total RNA was extracted using the QIAGEN RNeasy mini kit and DNase treatment was done after RNA extraction using the PROMEGA RQ1 RNase-free DNase.
Does anybody know why I am getting this atypical RNA quality results? Thank you very much!
Relevant answer
Answer
Thank you very I much for your insights Dr. Fathi Karouia. At first I ruled out the RNA degradation because of the good clean bands when running a denaturing gel, and RNA quantity with Qubit was really good. So I suspected that the atypical baseline was due to contamination. I did perform the experiment a second time for a new RNA extraction. However, instead of using the PROMEGA DNase after the RNA extraction I used a QIAGEN DNase compatible with the RNeasy mini kit for a in-column DNA digestion. It work perfectly and I got a high quality RNA this way. So I am curious to see if any other researcher ever experienced problems with DNase treatment after RNA extraction for downstream applications?
  • asked a question related to RNA Isolation
Question
1 answer
I've been trying to isolate total RNA from guinea pig's knee cartilage. I've tried Qiagen RNeasy kit, miRNeasy kit, trizol method with lots of modifications and have also used the RNaqueous total RNA isolation kit from Thermo Scientific but I've not been able to achieve RNA ratio of more than 1.4 for cartilage and 1.6 for synovium. In addition to the integrity the RNA conc. is also very low because the amount of cartilage available from the knee of guinea pig is very less (<15mg).
I homogenise the samples using Bead beater (bead mill).
Relevant answer
Answer
Hi Dikshit..because your starting material is less so, in Trizol method u can do overnight precipitation of RNA in isopropanol at -80°C as it will improve quantity as well as quality.
Hope it will work.
Thank u.
  • asked a question related to RNA Isolation
Question
2 answers
I am doing silica magnetic beads-based RNA isolation. I am facing some impurity issues because of washing. So kindly share some best washing buffer composition of wash buffer for especially for RNA isolation.
Relevant answer