Questions related to RNA Extraction
To check the expression of JAK2, STAT3, SRC and indoleamine 2 and 3 dioxygenase genes in the blood samples of people who do not have any immunological or hematological diseases, is the best way to isolate PBMC and check gene expression in PBMC or Can this be done in whole blood?
I have been trying to isolate good quality from mouse spleen for a while, but my RIN scores are always around 7 or below. My workflow typically includes harvesting the spleen in sterile pbs, sectioning it into 500um slices, staining overnight (rpmi and 1% bsa + antibody), imaging, dissociating in pbs, performing FACS (sterile method), collecting the pellet (centrifuging sorted sample), then lysing in trizol for rna extraction. I know that there are many steps that could be affecting the quality, but I always make sure to include an unprocessed control that is dissociated in trizol immediately after sectioning, and still the RIN is not satisfactory. There was one time I was able to obtain a RIN of 8.3, but I am wondering if there is anything specific I need to do to get reproducibly high RIN scores. I think my processing steps are not affecting the quality that much since the RIN scores of my unprocessed sample and processed samples are usually in the same range. Any advice would be valuable!
Is there any affinity of microRNA to glass surfaces? I tried to homogenize brain samples in order to get maximum yield of microRNA.
I found only kits which purify genomic DNA and total RNA sequentially. I'm looking for a protocol for isolation of total RNA and plasmid DNA from the same sample in mammalian cells.
I'm optimising a new RNA extraction protocol from yeast. The RNA extraction with formamide works great (the yield of RNA mass/YDM is even better than our established protocol) but the final volume of formamide in which the cells are disrupted makes the sample very diluted. I was thinking of ethanol or LiCl precipitation, maybe using SpeedVac (although formamide boiling point at normal conditions is 210°C so I'm not sure how possible this is; for two-phase extraction the only common solvent it doesn't mix with is diethyl ether which does not dissolve nucleic acids well). As the formamide extraction protocols don't seem popular, the published sources are very limited. Does anyone have experience concentrating RNA in formamide?
I am currently working in the process of automating our research lab to be able to process a higher number of samples. Our work consists in detecting and quantifying pathogens in mammalian samples (e.g., blood, faeces, tissue, swabs). As such, I am interested in an automated extraction system which produces good DNA/RNA yields to be sent for NGS.
Until now, we have been using Qiagen kits for our manual extractions, so I thought that a machine from the same brand would do for us. However, I've been told that the QIAcube Connect does not really take that much work out of your hands, and that the sample volume obtained at the end of the process with the QIAcube HT is way lower than the one with the manual kit. I have also checked other machines, such as Thermo Scientific's Kingfisher Flex and its kits, but do not know how well they do in comparison with Qiagen's kits.
Based on your experience, which automated extraction system would you recommend? And which brand of kits have you used with it? The system and kits do not need to be from the brands mentioned here (as long as the produce good results).
Thank you very much in advance.
I'm currently working with an organism which has no established endogenous control mirna that I could use to normalize qpcr data. I'm thinking about instead using an exogeous control mirna such as the ce-39 mirna to normalize quantitative data. I'm thinking it would be best for my purposes to spike it in before rt/cdna synthesis but I also see many papers where it's spiked in during the rna isolation step.
So I suppose I have two questions:
1) Is it valid for me to use an exogenous control to normalize qpcr data when I have no real other option?
2) Can I simply put it in before the cdna synthesis step and then compare the mirna results of my gene of interest to it? Or do I need to put it in during the rna isolation step?
I have seen it mostly as an rna isolation spike-in but this was also when they couldn't control for the amount of rna being put into the cdna synthesis, whereas I am able to obtain enough rna (I'm not using plasma/serum, which is known to be difficult to obtain mirna from) to control for the amount being put into cdna synthesis.
I isolated cells using magnetic beads, but I didn't remove the beads from the complex (sample-antibody-beads). They are still linked. However, I would like to know if is possible to extract the RNA from these cells while still coated with dynabeads (magnetic beads). Does someone have any clue/idea about the possibility to do this without compromise my sample?
My concentration was 123.5 ng/uL
260/280 Ratio - 1.725
260/230 Ratio - 0.401
Can I synthesize cDNA from the above RNA?
Need suggestions please.
Last week I found myself researching how different storage of cells in RLT buffer might affect the end results and found many different answers. Some say it is best to snap freeze the cells on dry ice, other say that room temperature is fine. Others mean that storage of cells on dry ice will severely affect the recovered yield. With all those different answers in mind I did a little test and thought id share it with the network.
I used equal amounts of BR5 mouse cells for each sample and then did different isolation methods, namely;
1. collect in lysis buffer, isolate RNA immediately, measure RNA, freeze on dry ice for 1h, measure concentration again
2. collect in lysis buffer, incubate in RT for 2h, isolate RNA & measure
3. collect in lysis buffer, incubate on ice for 2h, isolate RNA & measure
4. collect in lysis buffer, incubate on dry ice for 2h, isolate RNA & measure
I found that there is no significant difference in RNA yield between the treatments, on the short term. So, if you forget your samples in RT, you should be just fine!
Attached is a more detailed presentation of my findings
I wanted to perform RNA extraction of U937 cells (Human Myeloid Lymphoma) by TRIZOL method, after adding TRIZOL to the sample I had kept it for incubation at room temp. and since I was not able to Extract RNA immediately I thought that I will keep it at -80C however I forgot to keep the samples at -80 before leaving. I wanted to know If I should proceed with RNA extraction as It will 2 days that the sample will be in trizol and after which I can process it as I am not able to find any article related to this.
I wanted to perform RNA extraction of U937 cells (Human Myeloid Lymphoma) by TRIZOL method, after adding TRIZOL to the sample I had kept it for incubation at room temp. and since I was not able to Extract RNA immediately I thought that I will keep it at -80C however I forgot to keep the samples at -80 before leaving. I wanted to know If I should proceed with RNA extraction as It will 2 days that the sample will be in trizol and after which I can process it.
For MNV preparation, I inoculated MNV in Raw 267.4 cells (70 confluence) in a 6-well plate for two days, after observe CPE, I froze 6-well plate to -80C fridge for 1h and thawed at 37C water bath for 2min. This freeze-thaw cycle were conducted 3 times. then I collected supernatant in 6-well plate and centrifuged. I collected cell supernatant again to stock at -80C until use.
For MNV RNA extraction, I used QIAamp Viral RNA MINI Kit. I followed the protocol in the kit, added 140ul MNV stock to and used tips with filter. Before experiment, I spread RNase Away to clean the bench. All procedures were conducted on the bench.
After I extracted RNA, I used nanodrop to test concentration and purity. The concentration of my samples was about 50ng/ul, but the number of A260/A230 ranged from 1.16 to 0.49, much lower than 1.8. I'm not sure the most possible reason may cause contamination in my samples, or during my process.
if my MNV stock concentration is too low?
if my working environment is not clean enough?
or some hints for operation on RNA?
We want to perform a human RNA extraction from cell culture for an RNA-seq, but we have a viral RNA extraction kit (Quick-RNA™ Viral Kit-Zymo research) available. Therefore, we want to know if any methodological issues can interfere with the results if we use the viral kit.
I am trying to understand whether it is important for me to use an RNA Cleanup kit (such as NEB Monarch #T2030S).
I am isolating microbial RNA from an environmental liquid sample, and I subject this RNA to subsequent DNAse I treatment. Does it really make a difference if I do not clean up the inactivated DNAse and buffer salts, and just go on with RT/qPCR? I am guessing it should work without a cleanup, but does such a cleanup help in some ways that I might be missing? What is your experience?
I have got a good yield of RNA from my tissue samples (chicken intestines) and my 260/280 ratio is the in range of 2.0 - 2.09. However, the 260/230 ratio is appreciably low, in the range of 0.2 - 1.5. Can somebody please suggest a solution? My tissue samples were frozen in RNAlater. RNA extraction was done using Qiagen RNeasy mini kit.
Does a low 260/230 ratio affect downstream applications i.e cDNA synthesis and qRT-PCR?
I isolated RNA from fish intestines, and here are my results. The pictures were my two attempts. I already tried lowering the RNA concentration (<1000 ng/ul) by diluting it with nuclease-free water. Apparently, not many published papers did fish intestine RNA extraction, so I can't find a reference that would help me understand my result or if I did something wrong. I did RNA extraction of the kidney, liver, and spleen from the same fish before, and they were okay. So I think the intestine needs an additional step to get the three 28S, 18S, and 5S rRNA bands? The gel electrophoresis was done using 1% agarose, 60V for 20-30 min. I also added FluoroVue to the gel, and the samples were preserved with Trizol at -20 or -80. I used 10kb ladders. Any insight, advice, or suggestions are appreciated. Thanks!
in trizole method RNA extraction the smashed sample with trizole was preserved in normal freeze (-4) for 5 hours then bcp was added. will i be able to get results??
I need to do RNA extractions of a large number of samples from developing wheat grain, and plan to do them in the batches of 12. Is is sensible to keep the homogenised samples (with pestle and mortar using liquid nitrogen) on dry ice whilst the entire batch is homogenised before proceeding to the extraction protocol? Keeping in liquid nitrogen would probably be best but is dry ice also safer? The first/foremost sample will have to be on dry ice for roughly 1-hour by the time all 12 are grinded.
I'm looking for RNA extraction kit for isolation from cultured cells. I'm working with neuronal cell culture (SH-SY5Y).
Thank you in advance for your suggestions.
I performed a qPCR to assess the collagen type 1 gene expression in the mesenchymal cell line.
prior to the test, I got a little amount of total RNA than what I regularly get (10 ng/ul) as the cell growth was low.
so during performing the qPCR I found out that my housekeeping gene has a Ct of about 30 but the target gene (collagen 1) has no Ct in 40 cycles. so I decided to extend the cycle number to 60 cycles. I observed the Ct for collagen type 1 on cycle 50.
if there were no primer dimer and nonspecific products in the result, is this result reliable for gene expression assessment?
thank you for your kind guidance in advance.
I am doing RNA extraction using trigent (trizol equivalent ) , when I precipitate the RNA by iso-propanol I had a semiliquid very transparent colorless layer at the bottom. Is this the RNA pellet? or I need to do further steps to get a semi white pellet?
Thank you in advance
I am trying to find a protocol for cDNA library preparation. The problem is that we have to extract RNA from a very low volume of plasma/serum (less than 1mL. Since we are only using 1mL of blood, the starting volume of plasma/serum will probably be in the ballpark of 500 uL). We usually use NEB library prep and Qiagen for RNA isolation. With the latter, we get a decent yield but we need at least 3mL of input serum/plasma. Since most kits require at least 100 ug of input RNA for library preparation (we are trying to aim for 100 ng), we are trying to find a protocol that will allow us to use very low RNA sample input (concentration) to get a decent read without high adaptor dimer formation. In addition, any recommendation on the extraction of RNA from a low volume of plasma/serum is highly appreciated.
I'm going to make a phasemaker tube for RNA extraction like Thermo fisher Co.
Which polymer do you know has a density more than chloroform but lower than phenol?
mRNA is said to be a very unstable species of RNA which degrades rapidly making it difficult to isolate a significant amount of it from cells. However, RNA extraction protocols use protein denaturation buffers which potentially denature RNase enzymes, so what could be the cause of mRNA degradation in these extraction processes?
I've been having some trouble isolating bacterial RNA from a gram positive organism for a RNA Seq analysis. My problem is that I always get a very intense "cloud band" on the agarose gel around the position where the 5S RNA band should be.. I've tried several protocols and kits, with and without bead beating, Trizol, Lysozyme, but it happens every time.. The first idea was that these are products of degradation, but then again the intensity of the 23S and the 16S bands clearly remains very high. And also, on a Bioanalyzer this 5S band definitely does not look like degradation, but rather as a sharp peak around 127 nt.. Does anyone have any experience with that? If this is in fact the 5S rRNA, why do I get such accumulation, how should I get rid of it and would it temper with my RNA Seq results?
Thank you all in advance!
When I try to perform an RNA extraction with the RNeasy Qiagen kit on my M. abscessus bacterial cultures grown in the presence of kanamycin or amikacin, the RNA concentrations obtained are very low and the ratios 260/230 very low. I noticed that following the centrifugation of my cultures, my bacterial pellets appear dark orange while the antibiotic stock does not show any staining. Washing my pellets with PBS does not allow me to get rid of this coloration. After the mechanical lysis of my bacteria, and the passage on column, I notice on the filter a deposit of the same orange color. I can’t find an explanation, and I don’t know how to solve my problem. If any of you have suggestions, I’m willing. Thank you very much for your help.
I'm about to extract HBV-DNA from plasma. I have modified some homemade methods that are regularly used for Human genome extraction. But I could not get enough DNA for positive plasma samples. I also had a problem with a high concentration of proteins in plasma. I tried a high concentration of NaCl (6M), but it couldn't precipitate even 50% of proteins. I just have seen this problem with serum samples. Maybe it is because of the high protein concentration of plasma. By the way, I also don't want to use the phenol/chloroform method.
Who can help me?
Thank you for your consideration in advance
I'm working on an expression gene project for Human Ovum. I have six ova for each sample and have to extract RNA for cDNA synthesis. Can I go ahead with the conventional phenol/chloroform method for RNA extraction? Do I get enough RNA to assess gene expression ?
Thank you for considering my question
As the capsid of viruses is mainly made of proteins, unlike animal cell membranes, can I still use the SDS lysis ( SDS, NaCl, Tris, KCl, MgCl2, EDTA) buffer for viral DNA extraction?
Do you know any protocol for none-column based Viral DNA extraction?
Is it ok to use phenol/chloroform precipitation?
I really appreciate any help you can provide.
I'm working on a set-up for COVID-19 RNA extraction, and Sodium citrate is needed in the protocol for lysis buffer and precipitation buffer. Do you know any alternative for sodium citrate that can be applied in COVID-19 RNA extraction?
I am having a hard time solubilizing proteins or extracting protein in general from the Zymo Research RNA miniprep kit (Guanidinium-based buffer). I am planning on extracting both the RNA (for RT-PCR) and protein (for Western blots). For the protein fraction, the kit specifies to precipitate proteins using acetone. And I had no luck solubilizing the precipitate. I also tried TCA precipitation with acetone washes but the pellet won't solubilize in RIPA. I also tried resolubilizing directly in loading buffer but got no luck still.
Is there a different way to extract proteins from a Zymo RNA miniprep kit fraction (like buffer exchange with a buffer compatible with western and BCA assay)? Or, how can I solubilize the precipitate?
Hi, I've previously used the Qiagen RNeasy Micro kit for RNA extractions and got great results for primary neurons and oligodendrocytes. I've recently switched to the Meridian Biosciences Micro Kit which is the same concept, although uses TCEP instead of BME as a reducing agent with lysis buffer. I got next to no RNA and terrible ratios (less than 5ng/ul). I lyse my cells on ice with the RLY lysis buffer and TCEP combo before putting them in -80 to freeze. Would not snap freezing the cells in liquid nitrogen be the issue? I would appreciate any help!
I have been having some issues with extracting RNA from A viteae L3s , the TRIZOL method doesn't seem to work very well, and I have been trying different speeds/times with the TissueLyser and beads from QIAGEN, and freeze/thaw cycles as it seems that cuticles are very hard to break. I recently tried the RNeasy fibrous kit as well, with less beating, but the RNA quantity is so low <10 ng/uL (nanospec read).
I always try to extract them (A. viteae L3s) along with some controls (like B. malayi L3 or B. pahangi), every time I change something in the protocols. The controls always work..
They come in 1X PBS and usually the number is about n ~ 500... As cuticles are rich in collagen, I was thinking of applying a treatment maybe with collagenase or if it's full of lipids to precipitate them with acetone ?
I am also afraid that too much processing might be shearing the RNA as well....
It has been a couple of months since I've been struggling with it so any similar experience/troubleshooting might be really helpful ! Thank you !!
Phenol - Chloroform based RNA extraction methods are most widely used for RNA extraction. I am wondering if people have tried alternate methods for cell lysis (yeast, animal cells, plants cell, etc), specifically using SDS and proteinaseK ? The idea is to avoid phase separation-based methods and toxic organic solvents like phenol.
- What kinds of buffers can be used for lysis?
- How does one get rid of SDS and other chaotropic agents used during cell lysis?
Thanks for your valuable insights.
Dears, I am doing qpcr of genes with cdna made of RNA from adipose tissue. In the efficiency curve of the primers, the cdna serial dilution does not promote adequate amplification. The less diluted points dots are having less amplification. Example: in the photo, the last amplification is the highest dilution (1:2). I believe it is the presence of an inhibitor. Has anyone dealt with this? Know what to do? The 260/280 and 260/230 ratios are fine. The profile on the electrofluoresis gel is also good. The RNA was extracted with silica glass beads (sterilized and washed with acid), Trizol according to the manufacturer, and then the material was washed with precipitation with 3M sodium acetate and 100% ethanol. Resuspended in nuclease-free milliQ water.
I'm extracting RNA from fiddler crab (Leptuca pugilator) larvae and embryos for Tag-Seq. I've extracted using an RNAqueous RNA Extraction Kit and we wanted to determine RNA quality and integrity using the Agilent 2100 Bioanalyzer System.
I'm attaching screen grabs of the gel and peaks produced by the bioanalyzer. I expected to see 28S and 18S bands and peaks. However, I have one larger band on the gel and one large peak that seems to lie between 28S and 18S.
Has anyone seen anything similar using this instrument? Any insight would be highly appreciated. I would love to hear from someone that's worked with a similar species if y'all are out there!
I am running RNA extractions on whole gut samples for downstream RNAseq. For one individual I realized there was a length of gut tissue still in the original collection tube that I didn't add to the homogenization solution. I'm not sure what region of the gut it actually is or proportionally how large it is relative tissue that was homogenized (it is smaller), but I'm worried that if there are regional differences in RNA expression profiles that will bias the RNAseq data towards the already-processed portion of the gut.
Is this sample salvageable? If I extract RNA from the leftover tissue, could I just combine the total RNA sample volumes from both prior to sending in for sequencing? Alternatively, if we sequence both separately could we normalize and combine reads somehow? Are there any other strategies that would be more robust to prevent bias? Thanks in advance.
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.
Hello! I've been extracting RNA from paraffin blocks using Qiagen's RNeasy FFPE kit and looking at DV200 to assess their level of fragmentation (this is to determine whether or not that tissue block is usable for downstream experiments).
For my first two rounds of RNA extractions, most of my tissues came back with DV200 > 50% (which is what we wanted). However, all of my RNA samples following the 3rd round of extraction (I've done around 10 rounds of RNA extraction since), came back with DV200 < 50% (very poor quality, high level of fragmentation). Among these are RNA from the same paraffin blocks which I've previously tested in my first 2 rounds of RNA extraction and had good DV200 value i.e. > 50%. I suspected something happened between my 2nd and 3rd round of RNA extraction but I couldn't figure out what. It's always been the same individual doing the tissue cutting/paraffin curl collection and RNA extraction. Has anyone ever encountered this problem before and could advise some things that I could do to improve my DV200 results? (Since I HAVE gotten good DV200 results before for some of my blocks, I reckon this is more the case of poor RNA extraction technique than the quality of the block itself.)
Thank you beforehand for your help!
I am looking to send some RNA samples off for sequencing.
I have used a Trizol-Chloroform method for extraction on Gram positive bacteria that have been harvested and the pellet stored in RNA protect.
But I am getting lots of RNA degradation when ran on a gel, but TOO HIGH out of range when assessed on the Qubit BR RNA kit.
Would these still be okay for sequencing?
I sent off some samples for RNA seq and got a fail on their QC :(
I extracted using Trizol/chloroform.
I will attach the gel image they sent vs the gel image I have as well. Would it be okay to send off for sequencing as is?
Or should I clean up the extract?
If so, is there a protocol to remove DNA from RNA? Maybe DNase?
After having unsuccessful RNA extractions doing it myself the first time, and having the impeding time pressure of final year of my PhD, I am looking to send off some bacterial samples to novogene to have the RNA extracted from them for RNA seq.
I want to use RNAProtect to stabilise the RNA so that minimal degradation occurs.
How do I use it? I have read that it is generally 2x volume of culture, but I am working with 100ml cultures... Would there be a way to minimise RNAprotect consumption? Eg. Could I pellet a 100ml culture, resuspend in 10ml, for example, and then only use 20ml RNAProtect?
Thank you in advance
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?
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.
Since heparin inhibits reverse transcriptase, heparinase I treatments prior to qRT-PCRs are recommended. NGS also uses reverse transcriptase, so I was wondering whether anyone has tried doing a heparinase treatment of RNA samples prior to small RNA library preps? Is this a safe thing to do?
Currently I'm concerned with typical extraction of RNA from liquid samples in order to determine specific activity, i.e. detect active transcription of a few target genes. Since sample volumes are rather significant, I am looking to minimize costs of RNA stabilization. Fortunately, in the current setup I only need to stabilize the sample for a duration of ca. 30-40 minutes between sample withdrawal to RNA extraction bench. An opinion exists that for this amount of time, a stabilization solution is not even necessary, but it is difficult to assess this claim in the current setup for to several reasons not discussed here.
In any case, I do not need the *best* stabilizing solution, I just need a solution that will preserve the integrity of mRNA transcripts for downstream RT and PCR amplification for these 30 minutes. I know that supersaturated ammoniums sulfate solutions are used in similar situations, but it is also said that ammonium sulfate may interfere with downstream PCI-based RNA extractions. Maybe there is a simple way get rid of ammonium sulfate from the sample?
Any helps is very much appreciated :)
Best way for plasma separate for rna extraction (miRNA and mRNA)
incase there is no cool centrifuges
I'm doing RNA extraction with TRIZOL, but I just ran out. I saw some bottles identical to the TRIZOL so I assumed I had more but it turned out to be "buffer saturated phenol".
is this the same as TRIZOL? Can I use buffer saturated phenol with the same volume as I use TRIZOL?
If it isn't the same, is it possible to freeze the cell pellet so that I can do RNA ext when I can get more TRIZOL?
I'm doing RNA Extraction, Reverse Transcription and qPCR on synovium samples from mice that are dissected in half. I use Thermo-fisher RNAqueous Micro Kit for the RNA Extraction.
During RNA Extraction, I first remove the sample from -80 and immerse it in 100μL of lysis solution. then I homogenize the sample using the following mixture;
1. 10 ml PBS (without Calcium chloride and Magnesium chloride)
2. 1 tablet of protease inhibitor
3. 500μL Tween 20
then I add 1000μL of this mixture to my sample and use manual homogenizer to homogenize my tissue.
after that, I add 50μL of 100% ethanol to my samples and apply it to silica-based filter. then I centrifuge it at max speed for 30 sec.
then I wash it 3 times with 180μL wash solution 1 and 2 and centrifuge it for 10 sec after each wash. at the end, I centrifuge it for 1 min.
Then I replace the filter cartridge with new one and elute the sample in 7.5μL of elution buffer that is pre-heated to 75C. Incubate it for 1 min and spin-down for 30 sec. Then I repeat this step with another 7.5μL of elution buffer.
For Reverse transcription, I take 14.8μL of sample with 1μL of Oligo dT and 1μL of 10mM dNTP (16.8μL final volume)
then I place it in thermo-cycler for 5 min.
After that, I add 0.2μL of RNAase inhibitor, 1μL of M-MuLV Reverse Transcriptase and 2μL of 10x reverse transcriptase buffer to the sample and place it in thermocycler (37C for 50 min, 72C for 15 min, 4C infinite hold),
for qPCR, I dulcet the cDNA using 1:5 RNAse and DNAse free water, then further dilute it in RNAse and DNAse free water to get 1:50 dilution.
Then I add 4μL of diluted cDNA and 6μL of mixture including 0.6μL of forward and reverse RPLP0 primer (10mM concentration) and 5.5μL of 2x SYBR GREEN PCR Master Mix. and load my sample in 384-well plate and run it.
I tried changing my dilution to 1:25 instead of 1:50 but still don't have the result.
When I use cells (THP1) around 450,000 cells, Cq value is around 27.3 but for synovium is around 34. Still, both values are high for RPLP0 gene.
I use a silica column based RNA extraction kit which includes a guanidinium thiocyanate lysis buffer. The kit was very good and I always got pure RNA with A260/280 higher that 2 and A260/230 higher than 3!! (first figure)
However, after a while -using the same sample- after RNA extraction randomly the A230 becomes negative! and accordingly A260/230 gets negative, too (second figure) .
The blank is the same Elution buffer. And I'm wondering what could be the reason!??
I have my blood in Paxgene RNA tubes. I'm quantifying with nanodrop and the kit handbook is not clear about the blank to used for nanodrop (to do a dilution in tris-hcl 10mM is what they recommend in the some proportion for the sample and the elution buffer (blank); but the results are similar and I think more correct if I just used the elution buffer to do the blank.)
My ratio 260/230 are about 0,33-0,68 , really low. I want to use it for RNAseq
Thanks in advance,
I have jellyfish samples (gonads and tentacles) preserved in ethanol and stored at -80º for about 2 years. I would like to know if I can use these samples to extract RNA for transcriptomics.
Thank you all in advance!
Hi, may i know if anyone has experience in extracting RNA from oil palm kernels before? I have been trying to use different methods to conduct the extraction but none of the methods work till now. I have low yield, low concentration (almost none) and low purity of the total RNA at the end of the experiment (measured with Nanodrop). Can anyone please advise me on this? Thank you
I did gene expression analysis for a gene that is highly expressed under fungal diseases. The housekeeping gene did give ct values in the case of control and some treatments. I have read that I can take the average of all housekeeping genes and replace the missing with the average.
do you have any suggestions?
Can I estimated gene expression values without housekeeping gene
After and good RNA extraction with 260/280 and 260/230 ratios around 1.9 and 2.1 for both. I then applied a DNAse protocol to remove traces genomic DNA. My 260/280 ratios have remained unchanged but for a bunch of the samples the ratio 260/230 has decreased to around 1.0 to 1.45. The DNAse treatment has DNase buffer, DNase, and finally EDTA, some of those components are disturbing now.
How can I improve the 260/230 ratio and thus move to the next step of cDNA synthesis.
Thanks to anyone who can help
In single cell droplet sequencing, 2 cell lysis buffer are often chosen: 0.5%CA-630, or 0.2% sarkosyl 160 + 6 % of the Ficoll PM 400. What is the difference of these 2 choice in RNA yielding, mRNA completence and etc.?
Even though we have tried denaturing and non-denaturing gels, different kits for extracting, TAE or TBE buffer, different samples, and more, we have not yet seen two separate bands. There was only one band on the gel for all the samples.
Blood samples will be taken from remote areas. Blood will be collected in blood EDTAT tube not paxgene blood RNA tube. So we want to store blood samples and transport to lab for SARS-CoV-2 RNA extraction. Is it okay we isolate the plasma from whole blood on the same day of blood draw and then store the plasma at -80 for RNA extraction?
I will be grateful if any one could give a detailed description on their experience/precautions to be taken with RNA extraction from breast tissue (stored in -80). I am planning to use Qiagen RNeasy mini kit and homogenize the tissue with help of crushing in mortor and pestle with liquid nitrogen followed by homogenizing with lysis buffer. I was wondering the normal breast tissue which is usually with fat, will homogenize in the same way? Any information related to this will be greatly appreciated.
We are going to do an RNA-seq analysis to study transcriptomes\ profile of different organs in terrestrial orchid species within genera including Dactylorhiza, Ophrys, Himantoglossum, and Orchis but their underground fleshy tubers contain high content of glucomannan (a carbohydrate which gives special rheological features to products obtained from Salep) and it's difficult to obtain a pure RNA in presence of such contaminations. Is there any special and home-developed protocol to extract a pure RNA sample suitable for RNA-seq analysis from such tissues?
I separated mice serum by leaving it at room temperature for aroun 1 to 2 hours and preserved these samples at -30 for a month and then I transferred into -80. Now i intend to measure microRNA, but i am not sure if the RNA is undamged or it has been degraded!!! people always ask me to be aware while working in RNA since it’s highly sensetive!! so what do you think?
During my extractions the purity of RNA 260/280 is not too high. So I'd like to know if there's anyway to improve purity. I make quantification with Eppendorf BioSpectrometer.
I am doing a trizol based RNA extraction on a knee joint sample. My 260/28o ratio is anywhere from 1.85 -1.95 for most of my samples, but my 260/230 ratio seems quite low (1.08-1.2). My RNA concentration also reads anywhere from 1800 ng/uL - 4000 ng/uL... I dont even know if that is possible. Does that mean my sample is super contaminated? What can I do to improve this? I need to run RTPCR on these samples eventually. Thanks for your help!
I need to extract RNA from white blood cells (buffy coat layer) in human blood samples to do further RT-PCR studies. I am using Monarch's Total RNA Miniprep kit; however, the protocol card gives two a Mammalian Whole Blood option and a Tissue/ Leukocytes option. If I extract RNA from whole blood, what cells is the RNA coming from? Leukocytes, immature RBCs, etc?
If the RNA is from the WBCs, we would be able to do extraction directly on the whole blood sample.
I am currently centrifuging whole blood from a lavender top tube to separate out the plasma, buffy coat, and RBC layers. Then I use a pipette to suction off the plasma before removing the buffy coat and putting it in an Eppendorf tube. There is always lots of RBC in the sample. To get the RNA, I use the Monarch Total RNA Miniprep kit, but the 260/280 ratio is around 1.3-1.6 with 260/230 around 0.8.
Is there a better way to isolate the buffy coat and get more purified RNA?
Does anyone know about which carrier is better for RNA extraction: LPA (linear acrilamide), tRNA or glycogen? I need to analyze samples with low quantities of RNA, make c-DNA and Real time PCR. I heard that tRNA can inhibit c-DNA synthesis... is it true? maybe someone who has worked wtih these can help me! thanks!
I would like to extract Mycobacterium tuberculosis RNA from infected host cells. Is there a method that I can use. Aim is to study bacterial gene expression using RT-qPCR.
I will really appreciate your assistance
I am having problem with our microfluidic system for single-cell sequencing, that the cell cannot get lysed. Previous study ("Single-cell barcoding and sequencing using droplet microfluidics" https://www.nature.com/articles/nprot.2016.154) suggest using CA-630 in the Lysis-RT reagent and set the temperature of RT @ 50℃.
Is the cell lysed by CA-630, 50 degree or combination of both? I also tried having directly put cell in same concentration of CA-630 under 50 degree for same time yet cells are not lysed as well. How should I condition to realise cell lysing?