A few questions on RNA extraction and stability - (Oct/22/2005 )
I am a newbie grad student at a small lab. Unfortunately, I don't have very many people to answer my questions, as I am the only one besides my PI with any knowledge of molecular biology. I have experience with DNA, but not RNA.
Anyway, I am trying to get some RNA for real time PCR, and I am using a Strategene Absolutely RNA kit. I am using brain tissue, specifically striatum (as a positive control to validate my primers). After about 3 attempts, I finally managed to extract some RNA through my kit. That was about a month ago, and I had the following samples:
1. Conc = 1100 ug/ml
260/280 = 1.0016
2. Conc = 476 ug/ml
260/280 = 1.35
3. Conc = 1020 ug/ml
260/280 = 1.04
4. Conc = 619 ug/ml
260/280 = 1.22
I performed the same measurements about a month later (after a failed RT, which is another story) and found that I had lost RNA:
1. Conc = 150 ug/ml
260/280 = 1.9
2. Conc = 180 ug/ml
260/280 = 2.1
3. Conc = 110 ug/ml
260/280 = 2.6
4. Conc = 190 ug/ml
260/280 = 1.88
I know that it is recomended that you don't freeze and unfreeze RNA for fear of degredation, but I sure hope that this is not the normal result of 2-3 thawing cycles! Is this normal?
I had these samples stored in -80 for the whole time (with 2-3 thaws). Would adding RNA later help prevent this? RNA later is what basically enabled me to get the RNA to begin with, and now I am becoming scared to have my RNA away from it.
Also, it looks like I have a ton of protein contamination. Any ideas on how I can minimize protein contamination?
are you following the kit instructions to the letter? if not, where do you deviate? I have had excellent luck with that kit.
in what buffer are you resuspending your final RNA sample?
wow, your starting 260/280 is pretty scary. I do not understand why it goes down as degradation occurs...maybe there are proteases in your sample eating the contamination?
If you can get rid of the protein contamination and achieve good purity with your prep (your concentrations for adding sample to your RT cannot be normalized if all your samples have a random amount of protein with the RNA) I would recommend turning your RNA over into cDNA as soon as possible after isolating the RNA. cDNA is much more stable.
First of all, thanks for the reply
I am following the instructions to the letter as best as I can. The only real deviation is that I am using an electronic handheld tissue homogenizer (with individually plastic wrapped pestles (sp?)). For the previous (failed) extractions I had been using a manual homogenizer. I had to place it in DEPC to remove RNases; it was very difficult to get them out of solution, and I might have contaminated them with RNases in the process. I like the new one that I have. It is much easier to use, I feel more confident that I have properly homogenized the tissue, and I am sure that it is RNase free.
I am using the standard elution buffer, adding 50 ul at room temp (I know that the protocol says that I can increase yield if I warm it to 60 C, but I have not tried this yet). Right after spinning the tube in the centrifuge, I flash froze my sample on dry ice to minimize degredation. Do you know if the elution buffer has anything added to it to stabilize the RNA?
I want to do the cDNA conversion ASAP, but my RT enzyme is apparently denatured. I gave my PI a purchace order on Friday, and I can't do anything until I get more enzyme.
As for getting rid of my protein contamination, I don't know if there are any protocols for removing it from already isolated RNA, or if I should just try it again with a new sample.
With the sample that I have now, I think I will just try to RT one ul of my pure undiluted sample, and just use my DNA concentration to decide how much sample to add for QPCR. Does this sound good, or might I be adding too much RNA for a proper conversion?
i would not use the standard elution buffer which is probably contaminated by RNAses and would prefer DEPC treated miliQ. And for subsequen reactions, concentrated buffers are ok to get proper salts/pH for RT or whatever.
For your first OD measurements, i would say 1 you are poisoned by proteins or 2 your RNA are not resuspend properly. Try to heat sample before taking OD.
For removing proteins of "eluted" RNA, you should do a phenol (pH 4.7) chloroform IAA and then precipitate with ethanol (classical procedure).
I also get good results using that kit (with cells in culture).
You have a methodological problem with your measurement of ODs. Even if you had contaminating RNAses, yould would NOT expect differences in your measures over time. Degraded RNA absorbs UV just as intact RNA. Your 260/280 ratio should not be affected over time either.
My questions are:
-What OD values do you get? Too small absorbance values will give less precision.
-In what solution do you dilute your sample prior to reading absorbance? pH must be controlled. I suggest using Tris-NaCl-EDTA buffer (try to get the chapter on Nucleic Acids Quantification in Current protocols in Molecular Biology).
-I also strongly recommend having a RNA standard with known concentration (e.g. from commercial source), just to make sure your measument method is OK.
-I suppose you could re-extract your RNA with the same kit, with the risk of losing some material though. If so, please make sure you can trust your kit (buying a new kit may be worth the expense).
-Should you do phenol/chloroform/ethanol extraction, as suggested (though that would not be my first choice), be aware that phenol must not be oxidized, and that phenol MAY contaminate your samples at the end, lowering your 260/280 ration (and possibly inhibiting enzymatic reactions).
1st - heating the elution buffer to 60 actually does increase your yield (at least it works for me )
2nd - I don't really trust the elution buffer for storage; I find that my sample integrity remains a little better if I use either Fred's suggestion or carefully prepped RNAse-free Tris
3rd, (good point Serge!) What about your spec??
4th - I understand your samples are precious and difficult to replace, but in my experience a bad batch of RNA will not give you good qPCR results, no matter how hard you try to fix it up. I hope you can further purify yours and get good results.
for your RT, I would say do a calculation based on how much RNA you think you are adding. It's like PCR, you are limited by your dNTP's, primers, and template; when they are gone they are gone.
good luck. let us know if it works out for you
I'd definitely recommend RT ASAP, possibly the same day as RNA extraction (that's the way I have always done it). I've never had any problems with RNA if I reverse transcribed it the day I extracted it.
Also make sure that EVERYTHING you use is RNase-free/DEPC treated/handled with kid gloves...pipettors, table tops, everything should be wiped down with RNase inhibitor (I use Ambion wipes). RNase contamination is to be taken seriously.
I want to thank everyone for all of your responses. I have learned quite a bit. I will be ordering some new supplies that I hope will deal with this issue, including a new buffer to store my RNA in. Also from now on, I will do my RT within an hour of isolating my RNA.
Also one additional thing that I would like to note for anyone else looking through this thread with a similar problem. I called tech support to get their .02 as well, and they recomended that I double the number of salt buffer washes in order to get rid of excess protein, as that is apparently the protein removal step.
As for RNases, I am being as careful as I can. I have RNase free tips and microfuge tubes, and I spray everything else with RNase away.
As for my spec readings, for sample 1 before I got 0.698, and after I got 0.076 (the rest were similar, so I will not list them all), so it is not a problem with the reading being almost zero. That was the problem with my first three attempts .
I did just get in extra tissue, and I am content to just let this sample go. I will practice with non vital tissues that I have available, and apply the techniques that I have learned hear.
Thaks again everyone.
One more question:
If I use Tris, or a Tris-NaCl-EDTA solution, what pH should it be at? I am considering buying
this solution, that should end up with a pH of 8. Would this alkaline solution be good for storing my RNA? How about using it for spectrophotometry?
i'm currently changing DEPC water for formamide in case of RNA storage. But i can think to that due to the fact furthers experiments will be agarose or acrylamide gels... But i don't think it's suitable for RT PCR...