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Troubleshooting RNA Isolation

Isolating RNA is one of the more finicky protocols there is, and everyone who does it has their own personal tips and tricks to successfully isolate intact RNA from their samples with consistency. Although RNA can be somewhat unpredictable since it is so labile, there are a few common problems that occur which can be logically solved.

In my experience in helping people with RNA preps, these are the four main complaints when isolating RNA from tissues and cells. My solution to each problem is below. If you have your own tips, you’d like to share, please leave a comment.

1. Problem: Genomic DNA in the RNA
The RNA elutes with genomic DNA as evidenced by high molecular weight smearing or it appears clean on a gel but -RT controls amplify when PCR is performed.

Cause: No matter what method you use for RNA isolation, traces of DNA always carry through. This is true with TRIzol (phenol) preps and with silica spin filters. This can be caused by insufficient shearing of the genomic DNA during homogenization. If using phenol method, the pH of the phenol is key (it should be acidic) and your skill in pipetting only the aqueous phase will result in more or less DNA contamination.

Solution: The genomic DNA needs to be well homogenized so use a method that sufficiently breaks the DNA such as a high velocity bead beater or a polytron rotor stator. Samples will heat during homogenization but chilling samples in guanidine causes the salt to precipitate out. So you will need to balance the time of homogenization with time to cool down to room temperature.

The best way to remove the gDNA is with a DNase treatment, such as the RTS DNase™ kit, which contains a high activity, room temperature stable DNase that efficiently removes the contaminating DNA. Following DNA removal, a resin is used to remove the DNase enzyme without heat or EDTA. This type of kit is particularly recommended for samples rich in gDNA (ex. spleen tissue), samples that have been purified prior to DNase treatment, and when treatment of a partial sample is desired. “On-column” methods can also be used for samples containing low levels of gDNA contamination.

2. Problem: Degraded RNA/ low integrity
The rRNA bands appear smeared on a gel or the 18s is more intense than the 28s band. On the Agilent Bioanalyzer, you see a bigger 18s peak.

Cause: Degradation occurred at some point during processing. This can be difficult to pinpoint. It could be during collection and storage or it could be during extraction. It could also occur post isolation.

Solution: If the problem occurred during storage, make sure that samples are frozen immediately after collection. Use liquid nitrogen or -80C storage. For animal tissue, use RNALater and then store at -20C.

If the problem occurred during the extraction procedure, try adding beta-mercaptoethanol (BME) to the lysis buffer. Use 10 ul of 14.3 M BME per 1 ml of lysis buffer. BME will kill the RNases and help stabilize the sample during extraction.

If the sample is coming out of a freezer to be extracted and is not in a preservative solution, do not allow it to thaw. Homogenize it quickly in the presence of BME. Make sure not to leave behind any chunks of tissue. It needs to be completely lysed.

RNase degradation can also occur after isolation. Make sure that the water used for elution or resuspension of pellets is RNase free. Many kits provide water for RNA work that is DEPC treated or purified in other ways. More on the myths behind DEPC treatment and RNases can be read here.

In addition, 10 ways to work RNase free might also be a helpful article for you.

3. Problem: Inhibitors in the RNA
The RNA has an abnormally low 260/230 reading (below 1.0) or 260/280 reading or does not work in reverse transcription.

Cause: A low 260/230 in an RNA prep is indicative of guanidine salt carry over into the sample or organic inhibitors (such as humic acids or polysaccharides if the sample is environmental). Guanidine salts are used in TRIzol and in silica preps. These salts inactivate RNases, but will also inhibit proteins such as RT enzymes if present in the final RNA.  A low 260/280 measurement indicates protein contamination.

Solution: For low 260/230 readings, the best approach is to try more washes of the RNA sample. If this is a TRIzol precipitate, try washing it with ethanol to desalt it. For silica preps, a few extra washes with 70-80% ethanol should clear the column of salts. For samples already purified that are not clean, try ethanol precipitation of the RNA to desalt.

For other inhibiting compounds, such as humic acid and polysaccharides, you may need to re-purify the sample on another column and wash it well before elution. Some of these compounds will not be removed from RNA (or DNA) with conventional methods because they are too similar to the nucleic acids. In this case, you may want to consider using inhibitor removal technology for environmental samples.

A low 260/280 reading from protein carry over most likely occurred because of using too much sample for the kit or method. The sample overwhelmed the chemistry and the proteins were not completely removed.  Try cleaning up the sample with another round of your method- either phenol:chloroform and precipitate or add the binding salts and ethanol and bind to another silica spin filter. The protein should be easily removed. Next time, try using less sample and make sure homogenization is complete.

4. Problem: Low yields of RNA
The yield of RNA is lower than expected- either based on your previous results, or, based on reported yields for a certain tissue or cell type.  RNA yields can vary greatly between different cultured cell types and in different tissues. For blood RNA, it can vary from person to person.

Cause: If the yield of RNA is lower than you expected or know it should be, and the RNA is intact, so degradation is not the cause, then the homogenization may not have been complete. To isolate RNA, a strong lysis is key. Tissues stored in RNALater will tend to be a little more difficult to homogenize. Low yields could be caused by mistakes in weighing of tissue or in the cell counts for cultured cells. You may have less cells than you think. With blood RNA, the buffy coat can vary based on your skill in collecting the white cell layer and each individual patient.

Solution:For cases where the RNA yield is low but the RNA is intact, focus on the homogenization method and make sure you are getting good shearing of the genomic DNA and release of RNA from all cells.  If you see any pieces of tissue or debris in your homogenate, that is lost RNA.

It can be difficult to weigh small pieces of tissue so make sure you are using a scale accurate to the weight you need or you may see variation from prep to prep as well as other problems with purity and clogging columns when too much is used.  For cells, it is important to have an accurate count if you want to have consistency.

If the RNA looks degraded as well as the yield being low, the problem could be the storage or the homogenization may have been too hard and the sample was heated excessively for too long. Try homogenizing in bursts of 30-45 seconds with 30 seconds of rest.  Make sure to cut your tissue section and get it into cold guanidine lysis buffer (or TRIzol) quickly to prevent RNase activity during set up.

With silica spin filters, make sure the elution from the column is in enough volume to release the RNA from the membrane. More water will elute more RNA so use the largest volume that allows the RNA to be as concentrated as you need. Don’t try to use less volume than recommended by the manufacturer or you probably are leaving your RNA on the membrane. It is better to elute with more and concentrate after using ethanol precipitation, if maximal recovery is key.

And finally…

Isolation of RNA follows a similar protocol regardless of the sample source.  For all samples, homogenization is the first step and the most important step. A good homogenization needs to break cells quickly to inactivate RNases in the lysis buffer, and needs to break genomic DNA down in size to make removal more efficient.

Let us know what you think are the biggest issues in RNA preps and your tips and tricks used to overcome it. And if you have a problem now and want some advice, just let us know!

55 Comments

  1. Gabriella on March 5, 2018 at 7:50 am

    Hi,
    I was extracting RNA from bacteria using the RNeasy mini kit(50) but the results are not good. The concentration is too low. Its around 3.1-3.7ng/μl and absorbance of 1.43 to 1.58. The amount of RNA sample taken are 1.5ml, 3ml and 4.5ml each for both controls and treated. I have also done proper homogenization. What could the problem be? Should the amount of RNA sample taken be more? Can you please suggest

  2. maryam memar on January 1, 2018 at 9:19 pm

    hello
    I have a problem.: my rna is degraded in gel, but OD and concentration is good .I tried many things to solve it please guide me.

  3. Zhan Xuan on December 9, 2017 at 12:22 pm

    Hi Ms Susan,

    I’m extracting RNA from plants. I have store my samples at -80 degree but when i extract, the RNA degraded. I still confused as all my steps are correct. May i know which steps in extraction can cause RNA degrade?
    Besides is there any other way to extract RNA from root ? cause my root’s RNA degraded as i extract them. I’m using TRIzoL reagent.

  4. Atang on September 19, 2017 at 12:28 pm

    hei Susan

    I am extracting total RNA from blood, the tube i used immediately stabilize and lyse the cells upon collection. the RNA looks degraded on the gel, so i have planned to add beta mercaptoethanol directly to the blood after collection….anything you say on that??? i am using Tempus tubes and MagMax extraction kit.

  5. Lakshmi on May 18, 2017 at 12:04 pm

    Hi susan,
    can you help me to overcome problems associated with rna extraction from bivalve. I’m not getting a good yield while doing the trizol method and it is contaminated with protein

    thanks and regards,

    • Aldo on September 28, 2017 at 12:44 am

      I had low yield of total RNA. It seems like it was a binding problem. Concentration after The first flow through ethanol washing was significant higher >2000ng. I reloaded my Silyca column with the first flow through and I recover most of them. Could it be that other ionic molecules compite for the silyca beads?

  6. krishna on September 10, 2016 at 12:04 pm

    i stored Dnase I , RNase-free in -20s .
    after some times i remove form fregger
    how much time will capable to work it

  7. Lexa Chaudhary on August 8, 2016 at 12:14 pm

    Thank you for this tremendous article.

  8. Cham on June 29, 2016 at 2:52 pm

    Hi

    This article is very helpful. I work with plant material and have trouble isolating good quality RNA from Arabidopsis seeds. We use a Plant RNA isolation kit and it works great with seedlings but with seeds I always get very low concentrations as well as low A260/230 values. I do lot of qRTs and I end up getting nothing on my qRTs because of poor quality low conc. RNA. Do you have recommendations for plant seed RNA extraction? Any method to purify my already extracted RNA?
    Thanks so much

    • Amanda on March 7, 2018 at 2:51 am

      Did you ever get a reply? I’m having issues with my plant leaf rna isolations

  9. Poppy Redenström on June 28, 2016 at 10:16 am

    Hi,

    Thank you for a great article.

    I’m isolating microRNA using TRIzol but my 260/230 is below 1.0 . I have tried washing it with another round of ethanol 75% but I don’t get much difference. How can I get rid of the phenol contamination ?

    Thank you in advance

    • Bkeirns on September 8, 2017 at 3:14 am

      Did you ever figure this out? I believe I am having similar issues.

  10. Phd student on June 6, 2016 at 5:32 am

    Hi Sausan,
    I realize that I added the mercaptoethanol 100x less than it suposed. Do you think this will cause RNA degredation? meassured with nanodrop and the concentration seemed good as usual. However, I am wondering if it is confirm that RNA is intact?

    Looking forward to hear your thought,

    Best and Thanks

  11. sandra on May 31, 2016 at 4:40 am

    Hi Suzanne,

    I have a problem with my RNA isolation result using Trizol. After isoprophanol phase, I get the white pellet and wash it with ethanol 75%, then semi dry it for 15 minutes. Before running it on agarose gel, I check it with nanodrop and I got A 260/280 = 1.705, A 260/230=0.443 and the concentration is 695 ng/microliter. But, when I running it at agarose, there is no band at all? As an information I always apply RNA-se free water during isolation process.
    Could you please give me some explanation what is happen to my RNA?

    Thank you

  12. ludovica on April 21, 2016 at 9:52 am

    how can I clean the beads before the homogenization? if I do RNA isolation from tissue.

    • Dr Amanda Welch on April 21, 2016 at 3:39 pm

      You can acid wash them (google should have a protocol) or you could bake them. I would buy them RNase-free, though. It wasn’t too much more and well worth the time and hassle savings.

  13. behnoosh on December 19, 2015 at 1:50 pm

    hi
    I poored 1ml chloroform insted of 200 microlitre when i wanted to extract the RNA by mistake. by the way in the step of adding the ethanol 75% , i washed it twice. so please lead me what can be happen in this case for my RNA.

    THANK YOU BEFORE

    • Dr Amanda Welch on April 21, 2016 at 3:40 pm

      That shouldn’t be a big deal, but it would make your aqueous phase a bit harder to recover. In the future, if you put in too much chloroform you can add an equal amount of RNase-free, sterile water. Your RNA will be more dilute, but I never had a problem with quality or yield doing this. YMMV of course.

  14. Sudharsan on October 16, 2015 at 8:42 pm

    Hi Susan
    excellent overview! I am experiencing problems with rna extraction from blood and serum cultures. We are growing e.coli bacteria in neonatal blood and serum and use the cultures to extract the total rna. I used TRIZOL method. For blood, prior to TRIZOL method we used lysis solution to remove blood cells. The final yield of rna was very low ( blood= 87 ug/ml; serum = 24ug/ml). We had control (LB culture) and it had good yield (1040ug/ml).
    My question is is there any standard protocol where prior to RNA extraction, we can isolate the bacteria only ( devoid of any blood cells, Buffy coat). Prior to starting the TRIZOL method, after the PBS wash step, I had good pellets of both serum and blood ( blood pellet was hemorragic- even after i did two steps of washing). So, I was expecting a good concentration and was surprised for getting that low yield of total RNA. Any comments or suggestions will be appreciated. Thanks.

    • Amanda Welch on October 17, 2015 at 12:06 pm

      Were you able to get an idea of the density of your E.coli population prior to isolating the RNA? If your E. coli do not grow well in the neonatal blood and serum, then you’ll get a lower yield than in your LB culture (where E. coli grow like gangbusters).

    • Sonali on April 13, 2016 at 3:39 pm

      Hello!
      Did you have same number of E. Coli cells in your blood sample and LB control?
      Where were these samples kept, and for how long before processing?
      Since I will be doing a similar experiment, it would be great if you can tell me what worked for you?

  15. shuang on May 28, 2013 at 3:55 pm

    Dear Suzanne,

    Hi! In the last step of Trizol protocol, they suggest that after dissolve isolated RNA in water/TE, heat the sample to 50~60 Celsius degree fro 10~15minutes, do you know the reason behind that?

    Thank you so much!
    Shuang

    • Renu on December 8, 2015 at 1:50 am

      hi suzan can u tell me the reason for dissolving the rna at 50-60 degree celsius??

  16. zalenka on April 29, 2013 at 10:38 am

    Hi, Susan!
    Many thanks for the useful article.
    I`ve some trouble with homogenisation of the samples stored at RNA Later. We use Quiagen Tissuelyser LT with small metal beads (3-7mm) and homogenize human skin biopsies in RLT buffer with b-me. The problem is that total homogenisation of a sample (~3*3mm) takes us about 30-40 min of shaking with beads, and the RNA and gDNA obtained (we use Quiage kit for simultaneously isolation) seem to be degraded. The RNA bands are not very bright and seem to be blurry, and gDNA is not one line but a smear (although with the major heavy band). Skin biopsies are rather hard to homohenize, and it seems to me that RNA later makes it more firm. I thought maybe the reason of the degradation is long gomogenization without cooling? I would be thankful for any advice
    Alena

  17. nabiha on August 20, 2012 at 7:28 pm

    hi susan.

    Im a med student working on RNA extraction in the summer. Im supposed to do quantitative PCR(with sybrgreen) of the DNA after RNA extraction, but the RNA yields are always too low. i have been doing it for days but im not getting good results. i make sure everything is RNAse free but there must be something im doing wrong. hope you can help. thanks

  18. vishal319 on December 6, 2011 at 7:53 pm

    Hi Susan,
    I want to know whether I can recover the rna from the RNAlater, that is presnt in the RNA later because of the lysis of cells during storage. If yes , then how??

  19. allenandthomas on December 2, 2011 at 8:35 am

    Hi Suzanne:
    I am a newbie in mRNA isolation.
    I have a question to ask and hope you can help me out.
    I was using Trisure ( which is similar to trizol) to isolate mRNA from inslin producing cell ( which is differentiated from stem cell)
    Following are my steps:
    Collect cell( clusters) – add Trisure – grind the clusters – adding chloroform – centrifuge at 12000rpm for 15 min at 4 ºC
    Then here comes my question. I saw something weird( white stuff) are floating in the upper layer. Do you know what it might be and how to avoid it happened again. Does it mean that I didn’t homogenize clusters well?
    Thank you in advance.

    Allenandthomas

  20. cmdiez on November 30, 2011 at 7:50 pm

    Hi Susan,
    Thanks for your article, it’s very helpful and has given me some ideas about how to solve my problems with small RNA preparations.
    I’m isolating small RNA from total RNA using a 15% TBE-urea PAGE gel (following the small RNA Illumina protocol). I’m obtaining a high smallRNA concentration (around 200ng/ul) but the A260/280 reads are low (1.26-1.57) so I don’t know if I should go ahead with the construction of the library. Is there any method to purify my small RNA before start the libraries or should I extract total RNA again because probably I use too much tissue during the total RNA extraction?
    Any help would be greatly appreciated! Thanks in advance!

  21. maap.magni8626 on September 24, 2011 at 12:37 pm

    Dear Suzanne, how r u?

    Is RNAlater the same as DEPC-water? Or is it similar to Rnasezap or rnaseaway?

    Trizol is not very effective on small amounts of mammallian tissue (<50mg)
    But i know Trizol does not affect RNA yield and integrity. It just needs proper aseptic techniques

    • John Tadross on November 25, 2011 at 1:15 pm

      Dear MAAP

      RNA later, DEPC water, and RNA zap are very different things.

      RNA later is used to stabilise tissues, it’s a concentrated buffered salt solution which inhibits RNAse activity.
      DEPC inactivates RNAse in solutions and is used to treat water to ensure it’s RNAse free.
      RNAzap is used to remove RNAse from surfaces, it’s an alkaline reducing agent solution (DTT i think) which breaks the covalent bonds that maintain the RNAse catalytic site structure.

  22. Gaurav on September 22, 2011 at 11:58 am

    Hi Suzanne…
    thanks for such an informative article.
    when I isolate RNA from ticks with Trizol method, I am having some problems. when samples are run on a gel, I see faint 28s and 18s bands but a really thick and bright band at 5.8s (in most samples there isn’t any smearing). i really want to know if its degraded RNA or 5.8s band is like that only. if its degraded RNA can anyone suggest what should i do to avoid it? i already take most of the precautions and have new pipettes, filter tips, nuclease free tubes and reagents etc.

  23. Nasret on August 16, 2011 at 8:02 pm

    Hi Susan,
    I am having some problems with RNA extraction from mice tail tips. My concentrations are extremely low. I have been trying to find how much is typically extracted from mice tails but cannot seem to come across anything … My lowest concentration is 11.05 ng/ul, 260/280 is 1.54. Any advice would be greatly appreciated.

    Thanks!

  24. Poonam Tambe on February 24, 2011 at 11:44 am

    Hi
    Will u please tell me why acidic phenol (pH 4) is used in RNA isolation
    And the relation between number of cancer cells and volume of TRIzol reagent to be taken for RNA isolation?

  25. Suzanne on February 23, 2011 at 8:01 am

    Hi Elena,
    On your question, TRIzol is supposed to capture all of the small species. Just do not clean up the RNA on a spin column or it will wash away. Or, there are ways to enhance the binding of the small RNAs with the column.
    You need to add more ethanol in the binding step to a column. The more ethanol in the sample, the smaller the RNAs that can bind.
    But if you are using TRIzol, you should be getting it all.
    Suzanne

  26. Suzanne on February 23, 2011 at 7:58 am

    Hi,
    Make sure you are adding a salt for the precipitation- usually sodium acetate or sodium chloride. Chill the sample and then spin it for 30 minutes at full speed in the microcentrifuge tube to bring it down.
    You can add a carrier to help with recovery. Some people use tRNA or poly A RNA.

    Ambion has a product called GlycoBlue that helps precipitate the nucleic acids and gives the pellet a little color so you can see it easier.
    http://www.ambion.com/catalog/ProdGrp.html?fkProdGrp=137
    Maybe give this a try.

    Best,
    Suzanne

  27. SP on February 23, 2011 at 3:37 am

    I always landed myself to have low concentration of isolated RNA from the enterobacteriophages. I tried to keep isolating with greater amount, but no luck. All I can do to obtain a higher concentration is to pool all tubes and precipitates with isopropanol (I read that ethanol is working equally well for RNA precipitation), and elute with a smaller volume. However, despite all the precipitation and pooling, all I could have is still a quite low concentration. How I wish I could have more tips on how to concentrate the isolated RNA… thanks.

  28. Elena on February 16, 2011 at 7:11 am

    Thank you for the great article! Do you have any tips for increasing the micro RNA yield from cell samples? I use Trizol/isopropanol extraction for all my samples, but am wondering if it works for isolating micro RNAs as well as other types of RNA. Are there better methods out there for increasing micro RNA yield?

  29. Shell Frost on September 18, 2010 at 11:55 am

    For a qRT-PCR reaction I need about 1- 10ng of cDNA and for cDNA preparation I need 10-100ng. SO I have to dilute my RNA is DEPEC water. However, I am not sure as to whether I can store my diluted RNA and cDNA in the freezer or will it result in loss of integrity?

    Many thanks
    Shell frost

    • Suzanne on October 9, 2010 at 12:44 am

      Hi Shell,
      The cDNA will be stable in the freezer, but, it has been reported that nucleic acids stick to the walls of the plastic tube and there is loss. Some people add tRNA to help in the storage- presumably to block the binding to the wall. You may just want to quantitate it again if you’ve had it stored a long time.
      RNA is stable in water at -80C and if it is pure, it should be fine.

  30. Vonne Jak on July 6, 2010 at 10:05 pm

    Hello,

    I have phase separation problems after homogenization and chloroform addition. Some of the samples have a very thick interphase and little/no aqueous phase after centrifugation and once inverted aqueous phase. What will be the cause of this situation and how should I overcome this?

    Thanks in advance,
    Vonne

  31. Suzanne Kennedy on May 31, 2010 at 6:36 pm

    Hi Fulvio,
    I would try it in the RT buffer from whatever RT kit you are using. This way you can add it all into a reaction and not have inhibition. You should also set up some controls- maybe spike one of the single cell reactions with the purified RNA or some DNA of the gene and make sure it amplifies.
    And also do a single cell RT-PCR reaction with a high copy gene like actin so you know that you are getting detection of any RNA.

    Suzanne

  32. Fulvio Celsi on May 31, 2010 at 12:02 pm

    Hi Suzanne and HdM
    thanks for the suggestions…I’ve tried with plain water and nothin came out..I’ll try to use some detergents and also the temp increase.. I’ll let you know how it goes..! 🙂
    Fulvio

  33. will hock on May 29, 2010 at 7:47 am

    Hi fulvio!
    I have used “whole cell PCR” for regular PCR amplification of genomic DNA from gram positive and gram negative bacteria. Admittedly with small volumes of cell colonies scraped of with the tip of a pipette added directly to the PCR reaction and not single cells, however, it would seem that temperature increase is sufficient for cell lysis, in that PCR products were faithfully acquired with this procedure, for this application at least.
    hdm

  34. Suzanne Kennedy on May 28, 2010 at 2:49 pm

    Hi Fulvio,
    I don’t think water alone is enough to break the cell and release the RNA. I think usually there is a little detergent, such as triton x-100, to help solubilize the membrane. I think most Taq buffers have some triton or tween in them.

    I think what is feasible would be to put maybe the cell in 1X PCR buffer or 1X RT buffer (which I think contains some detergent), and then to heat it to lyse the cell, and then your sample is fully compatible with the enzyme.

    There are a few commercial kits on the market that work in this way also.

    This is theory- I haven’t tried this myself.

  35. Fulvio Celsi on May 28, 2010 at 8:57 am

    Hi Suzanne
    just a very stupid and basic question…so, if you need to have RNA from tissue, then there’s obviously the need of isolating it, but I’ve read papers where when challenged with RNA from single cell, they just put it into water and start with the RT reaction. Is this feasible? What you think about?
    many thanks!
    fulvio

  36. Suzanne Kennedy on May 15, 2010 at 1:17 am

    Thanks Eric!
    We used to recommend diluting before spinning it out of cultured cells when I was at Qiagen.
    Is it ok to dilute for cultured cells?
    Suzanne

    P.S. I like your avatar. Very cute rendition of you!

  37. Eric Lader on May 15, 2010 at 12:25 am

    Hi Pavan and Suzanne. I wouldn’t necessarily recommend diluting the RNAlater. One feature of the reagent is that if you wash it out (or dilute it), bad things can happen to the RNA in your sample. The inactivation of cellular RNase by RNAlater is about providing an inhibitory environment.

    One way to collect the floating stuff is to filter it all onto a membrane or something and then grab it and stick it into GiTC. Samples saturated in RNAlater can be plucked out and manipulated with no danger. You could even freeze the saturated tissue without the RNAlater.

    Boy this brings back memories. I haven’t worked with the stuff since my days at Ambion ten years ago.

  38. Suzanne Kennedy on May 15, 2010 at 12:16 am

    I will!!

  39. Pavan Kumar on May 14, 2010 at 5:07 pm

    Hi Suzanne,

    Thanks a lot for all the helpful suggestions. If you happen to talk to the ‘expert’ on RNA Later again please thank him for his great contribution.

  40. Suzanne Kennedy on May 14, 2010 at 3:43 pm

    Hi Pavan,
    I think you can dilute the RNALater- yes. RNase free water sounds perfect. That would change the density and the tissues should pellet. Some people have the same problem with cell cultures not pelleting and it needs to be diluted. That is the best approach.

    Best,
    Suzanne

  41. Pavan Kumar on May 14, 2010 at 12:04 pm

    Hi Suzaane,

    Thanks for helpful comment. I dissect out tiny tissues and keep them in RNA later (on ice). The entire procedure takes 1-2 hours after it I store the tissues in -80C. However when I thaw the tissue and try to pool them I still see it floating around in the RNA Later (even after centrifuging at 13k/ 1 min). I would like to collect these tissues (hundreds in number) from bottom of the tubes to pool them together. I was thinking to dilute RNA Later with nuclease free water to decrease viscosity but I am not sure how it will affect the RNA integrity. Any suggestions?

  42. Suzanne Kennedy on May 13, 2010 at 10:09 pm

    Hi Johnny and Pavan,
    I wasn’t sure of your answers and I have experienced floating tissue in RNALater as well, so I asked an expert (the inventor of RNALater) and here is what he told me:

    “If a small tissue fragment floats, just rotate it for a while (5 minutes) and it’ll be fine.

    Lots of samples get very hard in RNALater. RNAlater works by both drawing water out of the sample and causing a massive precipitation of proteins.”

    So that’s why samples get tough- but a good homogenization is all you need. Using a high speed bead beater, I haven’t had any problems getting normal yields of RNA from heart and muscle in RNALater- even stored up to a year at -20C.

    Johnny- what method are you using for the homogenization? What tissues give you the most trouble?

    Best,
    Suzanne

  43. Ken Doyle on May 13, 2010 at 6:54 pm

    Excellent overview! One way to avoid the problems with inhibitors like guanidine salts is to use a simple salting-out/precipitation protocol (originally developed for DNA, see Miller, S.A. et al. (1988) Nucl. Acids Res. 16, 1215). There are several commercial kits based on this method for purification of both DNA and RNA.

  44. Pavan Kumar on May 13, 2010 at 5:09 pm

    Hi Susan,

    Thanks for the helpful article. Another question regarding RNA Later.
    I am using it to store tiny tissues (which I would like to pool eventually to isolate small RNA population). However, I have observed that tissues float on RNA Later and pooling tissues from different tubes by centrifuging them is not possible due to high viscosity of RNA later. I would really appreciate your suggestions to solve this issue.

  45. Johnny Young on May 12, 2010 at 10:05 pm

    Hi Susan,

    I’ve encountered the troubles you mention in homogeneizing tissues stored in RNA Later. Do you have more information or references on that point? I’d greatly appreciate it!

    Thanks!

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