I have performed 3 RNA isolations on Jurkat cells using the phenol-chloroform principle, using Omega Bio-Tek RNA Solv® & following the protocol. I took care in not disturbing the separated phases when removing two thirds of the aqeous phases but the data I get when I measure the 260/280 & 260'/230nm absorbance ratios lead me to suspect contamination of the samples with phenol or other reagents.
Most of the samples have a 260/280 ratio below 1,6 and only one sample has a 260/230 ratio that comes close to 2 (1,84 to be exact). Subsequent cDNA synthesis & RT-qPCR have not resulted in genes of interest to show any fluoresence at all, only the housekeeping genes in 2 of 7 samples showed up. Problems with primers & RT-qPCR were ruled out as the same setup yielded viable results previously.
Thanks in advance!
absolutely, organic carry over will affect downstream analysis. temperature and handling will also affect RNA integrity
you could perform an RNA cleanup
https://cshprotocols.cshlp.org/content/2020/3/pdb.prot101717.full
I use 3M Sodium acetate pH 5.2, when precipitating in ethanol (step 3) it does really help to do this at -20C overnight. Air dry the pellet but just long enough to evaporate the ethanol, don't leave it too long. and then when RNA is resuspended in water to ensure there is some EDTA in there and then heat at 65C for 10 minutes to fully re-dissolve.
260/230 over 1.5 should be ok, i've gotten amplifcation on some with ratio of 1.3. I think degradation might be more of an issue. overmixing the samples during the isolation could degrade your sample. when I add the chloroform and shake vigorously - i do so by hand, do NOT use a vortex, this will shear RNA. also do not let the samples sit in the phenol for too long.
Zymo makes a nice Directozol RNAprep kit that is not too expensive and you can do on-column DNA digestion, it is pretty newbie friendly. Zymo will also give out samples of their kits for like 5-10 preps for you to try. You just need your own trizol.
https://www.zymoresearch.com/pages/direct-zol-rna-purification-kits
click on the free sample link at the top of the page (it takes more than 7 minutes as DNase digestion is 15 minutes, I can get RNA in less than 30 minutes)
good RNA isolation can be difficult to master so stick with it and just be patient.
Hello Melle Katerberg
I agree with Julie Ann Dougherty . RNA quality can be affected by impurities like phenol which can be carried over to samples. Residual phenol is assumed to inhibit PCR reactions by denaturation of enzymes such as polymerases and reverse transcriptase in a concentration dependent manner. So, try to use lower concentration of RNA than what you would be presently using.
You may want to refer to the articles attached below for more information.
Article Detection of phenol contamination in RNA samples and its imp...
Article Optimization of Phenol-Chloroform RNA Extraction
Best.
Thank you kindly for your in depth answer. I'm a biomedical research undergrad student and tips/advice like this from someone with more experience is always very useful.
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