Hey,
Usually, when I perform total RNA extraction, I directly synthesize cDNA and run qPCR without even analyzing or testing the purity of RNA. I am asked to use 15µl of RNA when synthesizing DNA. However, I am not able to understand the reason that is given to me, because in academia they taught us that RNA has to be tested for purity before proceeding with further experiments. I also don't understand the idea behind just using 15µl exactly to synthesize cDNA instead of calculating the exact amount needed. If there is logic behind that, can someone explain it to me in simple words?
Thank you
Talk with your advisor/supervisor. Did someone else already do the testing? Is this part of a really high-throughput project?
My advisor used to say "you can't rush quality".
Good luck!
Hi,
If you have a DNAse treatment step during the extraction process, verification of the purity of your RNA might be optional.
However, for best practice, it is always good to run an agarose gel that shows:
-Purity of RNA (no gDNA on the top of the gel)
-Quality of the RNA (2 discrete bands corresponding to rRNA), no smear of degradation.
The latest is especially important ave inconsistent qPCR results with low-quality RNA.
When I run RT-qPCR, I prefer to always use the same quantity (usually 1 ug of total RNA) rather than simply adding equal volumes of random quantities.
Good luck!
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