Checking the
quality of total RNA on an agarose gel.
- Use RNase free
reagents.
- Clean the comb and
gel box in RNase Zap.
- Prepare standard 1%
agarose gel (non-denaturing). Gel can contain ethidium bromide for
visualization of RNA or can be stained for RNA after electrophoresis.
- Load 1 ml aliquot of your
total RNA (if sufficient sample is available).
- Load a DNA ladder to
estimate the location of bands.
- Run the gel far
enough to get good separation of 28S and 18S ribosomal RNA bands.
- Take a photo with
good exposure that shows the wells down to the 5S ribosomal bands.
Notes:
•
The 28S and 18S ribosomal RNAs should be discrete without a
leading
smear.
•
The 28S band should be about twice as bright as the 18S band.
•
There should be little or no genomic DNA in or near the wells.
•
There should be no extra bands between the well and the 28S band. This is DNA contamination
• If
your total RNA samples meet the above criteria, proceed to the next
step:
spectrophotometry.
• If
your total RNA samples do not meet the criteria, contact CMG for
consultation. Samples may require
re-purification if there is obvious DNA contamination. If there is
evidence of
excessive degradation, consideration should be given to obtaining new
samples.