RNA integrity - (Feb/06/2009 )

i would like to check the integrity of my RNA that is isolated from a tissue sample, it is being converted to cDNA and used in qPCR reactions

somewhere i saw that you could check this by running it on a agarose gel, does this sound correct? what information can you gain from this? what would you expect to see and how would you know if your RNA had been degraded somehow?

thanks

dna_nerd on Feb 6 2009, 03:13 PM said:

that does help, but am unclear why you should only see a single band as it is total RNA from a tissue sample (mRNA, tRNA, etc...)

i assume these are all different sizes but maybe they are similar enough?

You need to run your RNA on an agarose gel to make sure your RNA quality is fine. Typically you should see two bright and distinct bands on the gel: 18s and 28s rRNA. If you see a smear, or weak bands, your RNA is probably degraded. Here are a bounch of protocols on how to run a agarose gel of RNA
http://www.protocol-online.org/prot/Molecu...esis/index.html (some links are outdated, but you can check the cached page)

thanks

Here's a gel photo, where you see DNA, 28S, 18S, and 5S/tRNA from top down.

In the past I used to run .5-1ug of RNA on 1.2% agraose Gels. You can typically see the 18s and 28s rRNA as has been mentioned. The smaller RNA were usually a faint band.

You can get a idea of your RNA quality (Band vs. severity of a smear), if contaminants are present, and a rough idea of RNA concentration. Even with a smear or faint bands in the RNA gel, I've gotten decent (~1kb) cDNA and PCR product.

all very helpful, thank you

looks like i could run a denaturing vs native gel, is a native gel good enough? would i need to heat the sample beforehand (if so, how much)?