Hello everyone,
I'm facing a problem regarding my pure extracted RNA samples, I extracted them from tissues of rats' livers and kidneys immersed in RNAlater, using the promega SV isolation system, and I dissolved the pure RNA in nuclease-free water and stored them at -80C right after the extraction.
However, there was a small accident where these pure samples had to be left at room temperature for almost a week or so. And so, I'm wondering: Are my samples of no use anymore? Shall I re-extract the RNA again or try measuring its purity and concentration?
I seriously have no idea to what I am supposed to do, and I really need your thoughts and advices.
Thank you so much in advance.
hi Najla,
I am afraid the RNA might be degraded. But you can verify by running a 2% agarose gel with EtBr, load 2-3 microgram RNA.
You should see intact RNA bands like this.
https://www.thermofisher.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/rna-protocol/agarose-gel-electrophoresis-of-rna.html
If you are lucky, RNA will be intact.
Goood luck,
Anand
Thank you so much Dr. Anand, I'll run the samples in gel as you recommended.
Thank you again.
You are welcome. If you see a smear, you might have to isolate new RNA.
Hi Najla,
Alternatively, you can run your RNA on the Agilent Bioanalyzer and get a higher sensitivity look at the electrophoretic profile. See the link below.
https://www.agilent.com/cs/library/applications/5989-1165EN.pdf
I am not optimistic after a week at room temp. If you can determine actual transcript size range, perhaps the samples may work in a targeted assay e.g. PCR targeting small amplimers.
Thank you Dr. William,
I actually extracted new samples of total RNA from my tissues in the RNAlater and compared them with the total RNA that was at room temperature via RT-PCR, they pretty much gave similar results which made me realize that it might held on at those circumstances.
thank you so much for the suggestion. I really appreciate your time.
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