Checking integrity of RNA

2015-01-13

azim58 - Checking integrity of RNA

Incubate one aliquot of 250-1000 ng of RNA at -80 C and one aliquot at 37
C.
Perform electropheresis with these aliquots on a 0.8% gel with a 1 kb
ladder. Run the gel at 220 V for 20 min.
If the RNA solution does not contain RNases or other contamination then
the 37 C sample should be about as bright as the -80 C sample. Also, if
the sample incubated at 37 C shows a lower 28S:18S ratio than the
control, the RNA may have contaminants. Two prominent bands should be
visible: 28S band around 4.5 kb and 18S band at 1.9 kb. The intensity
ratio of the 28S:18S band should be 1.5-2.5:1.

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I originally had this note below in the protocol, but it does not seem to
be accurate based on my experience from 2-24-13 in notebook.
Run the gel at 70 V or another voltage, but not too high. Do not use high
voltage in an attempt to avoid RNA degradation during electrophoresis
(http://www.evrogen.com/technologies/RNA-electrophoresis.shtml) <- this
does not seem to be accurate

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