Hi
I isolated RNA samples from cells and made cDNA.
In the case of troubleshooting, I would like to validate my RNA sample by looking at 18s and 28s.
but I don't know how to do it. I have searched the protocol but it didn't clearly show it up.
can you help me out?
Hello Jae Hyo Lee,
The most common method to check the integrity of your RNA samples is to run an aliquot of your samples on a denaturing agarose gel and later stain the gel with ethidium bromide. Sometimes you may have to use Sybrgold instead of the traditional ethidium bromide to stain the gel in order to increase the sensitivity.
The denaturing gel system is suggested because most RNA form extensive secondary structure via intramolecular base pairing, and this prevents it from migrating strictly according to its size.
If your RNA sample is intact, you will get a clear, sharp 28S and 18S rRNA bands for eukaryotic samples. The intensity of the two bands 28S:18S should be in the ratio 2:1 which is an indication that your RNA sample is intact. Partially degraded RNA will have a smeared appearance and will not show the 2:1 ratio as that seen for intact RNA. Completely degraded RNA will be seen as a low molecular weight smear.
For the protocol, you may refer to the link provided below.
https://barricklab.org/twiki/bin/view/Lab/DenaturingFormaldehydeGels
Best.
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