Good evening, everyone. I hope all is well
I want to ask about the validity of my samples, since they were Reserved as a whole blood since 2012 on EDTA tube (CBC tube with purple cap) at -20C and I want to detect gene expression by real time PCR on them now.
Are these samples valid for RNA extraction or the integrity of RNA was lost?
Also, if I want to detect C reactive protein in plasma by ELISA, Can I use these samples to separate plasma now or they were hemolyzed, and this protein was fractionated?
Finally, Is there a non expensive way to test the validity of this samples, as we were measured several markers on them in 2012 like (ALT, AST, Albumin,CBC, ETC....and compare it with its value now
Hello Wafaa Abdelaziz,
RNA and proteins are highly unstable biomolecules. Storing the samples even at ultra low temperatures does not help in such cases. Your blood samples are too old for any kind of RNA or protein detection. They could only be used for DNA extraction. You wont be able to do any kind of expression study on them.
I also agreed with Chitra. If you have separated the plasma at first instance then there may be a chance of doing protein analysis. But now your samples are too old to do those studies.
Usually the separation of plasma in EDTA-coated tubes is done the same day of blood collection. Additionally, samples must be directly stored at -80°C or in liquid nitrogen. I did not face such case before, but I think that by using these samples you risk to obtain false/noninterpretable results.
i agreed with both of them but at least try one or two sample and check integrity by finding RIN number.
all the best
Usually -20 is good for storing for short time (couple of months) 4 years is a long time for storing in -20. RNA and protein are unstable. For protein it was better to use serum (from Clot not EDTA blood). anyway concerning RNA you can try but not freeze and thaw and it is better to thaw once and slowly (on ice). Try one samples and after extraction of RNA run in a gel to check the quality. if you did not see the smear you can go forward for cDNA synthesis and doing simple PCR for house keeping. If it was ok you can go for real time (real time master mix is expensive and it is better to make sure).
If the cold chain was ok and there was no alteration in temperature it is possible to recover RNA.
Good luck and let me know when you succeed.
Very unlikely the sample will yield anything of value. If you took a gamble and dId analyze it, comparison to collection results from 2012 would tell you how degraded the sample was. However I agree with everyone else's advice. Unless your goal is to show that after four years stored at -20C the sample is no longer viable, I wouldn't waste the resources on testing.
Generally too old as others told. But you could still check the little chance for RNA. Go ahead extract the RNA, find someone who has a bioanalyzer that is Heber used in highly sensitive RNA assays like DNA EXPRESSION MICROARRAY. In conjunction with the nanodrop concentration result, you will be able to tell how bad your RNA is and the validity of using this RNA will be quantified and qualified. For protein assay, I am not sure.
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