Hi. I am just curious to ask.
The protocol of TRIzol states that the sample should be fresh or quickly frozen. So, if I process my "ethanol-preserved samples" through TRIzol, would it affect the total RNA extraction? OR... should I collect the fresh samples?
Hi Mirza,
I'm found the same question on Researchgate 4 years ago by Sarah Hemming.
This is the best answer by Rajkumar Sankaranarayanan:
If it is fixed with 95% EtOH, try re-hydrating the tissue with series of decreasing concentration of EtOH (say 95%, 70%, 50%, 30%, 10%) prepared using DEPC water and finally in DEPC water for 10 min interval at every step (time can be adjusted with the size of tissue). All steps should be performed at cold condition (an ice bucket will do the purpose). Then go ahead with TriZol method. The yield will be less, but you can fish up some RNAs.
Please check this:
https://www.google.com/amp/s/www.researchgate.net/post/Can_we_extract_RNA_from_95_ethanol_fixed_tissues/amp
Hope it Help.
For TRIzol based RNA extraction method to work optimally, you need fresh or frozen samples. However, if you are ready to compromise on the yield, you may use the sample stored in ethanol, but that will need pre-treatment i.e. bringing the sample back to its hydrated form. Use DEPC water, under cold conditions and in all cases avoid exposure to RNase.
Best wishes.
Thank you very much dear sir Mabrur Mabrur . It certainly helped alot.
If collecting fresh samples is not too hard, I would suggest you to use fresh tissues for RNA isolation. You should verify your RNA integrity once you extracted them either from freshly harvested sample or ethanol-preserved sample.
Yuan-Yeu Yau Thank you very much for the comment. How can I verify the integrity of RNA? by RT-PCR?
I have recently checked the Nanodrop Results of one of the vial. The conc. was 280 ng/ul and 260/280 was 1.15. It shows impurity but are these results give some hope for RT? (by RT i mean reverse transcriptase)
Thanks.
Jawwad Mirza You can check the integrity of RNA by loading it on agarose gel also. If the RNA has degraded, you will get smear. A quick google search will guide you how to make agarose gel for RNA quality check. There are always better and costlier ways to check integrity of RNA, but in my opinion, AGarose gel gives fairly good idea.
Bakhtiyar Alam Syed Thank you very much. The problem which I may face, as I faced for DNA, is I work with Mites (ACARI: ARACHNIDA). They are so small that even a very pure DNA extracted from 1 mite female (260/280 = 1.9) doesn't show any bands in 1.2% agarose unless i make a conventional PCR. I will make a gel run but I am afraid the DNA-story may repeat.
Jawwad Mirza In that case, you may either prepare cDNA from the RNA and set up a PCR. Preparing cDNA will make it stable for long storage. Or you may also set up a Reverse transcriptase PCR.
Best wishes
Dear Jawwad Mirza,
Sorry for the late reply.
For your first question: "Can an impure RNA sample shows some hope for RT?". This should be depending on your research goal. If you just want to do a simple RT-PCR for a specific gene, I think it should be ok. But, if you want to do RT-qPCR or a large transcriptome experiment, integrity and purity of RNA samples can affect your results. See paper below. What kind of RT experiment you are going to conunduct?
"Measurable impact of RNA quality on gene expression results from quantitative PCR" Article Measurable impact of RNA quality on gene expression results ...
Dear Jawwad Mirza,
For your second question: "How can I verify the integrity of RNA? by RT-PCR?"
1. You can run an agarose gel, as suggested by Bakhtiyar Alam Syed. Some people just run a 'native' agarose gel. However, it would be better if you can run a 'denaturing' agarose gel. See attached article. Because RNA can form many different secondary structures which affect its mobility in an electrical field if it is not maintained in a denatured state. Formaldehyde is used to keep the RNA denatured. However, as you predicted, I think your RNA would be too little to be seen on the gel.
2. If you can access to a Bioanalyzer, you can also use to check your RNA integrity. It gives you a so-called 'RIN' number. The higher the number, the better your RNA integrity (see attached figure).
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