Nov 23, 2020
Version 1
RNA Integrity Check V.1
This protocol is a draft, published without a DOI.
- 1In-house protocol
Protocol Citation: Elizabeth Fozo 2020. RNA Integrity Check. protocols.io https://protocols.io/view/rna-integrity-check-bpzgmp3wVersion created by Eadewunm
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 23, 2020
Last Modified: November 23, 2020
Protocol Integer ID: 44808
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Abstract
RNA Integrity Check: Modification of Protocol from Qiagen
Safety warnings
Formamide and formaldehyde are toxic and dangerous chemicals. Handle with care in the hood!
Before start
Pretreat gel containers and gel flask with RNase Zap
RNA Integrity Check
RNA Integrity Check
Determine the concentration of RNA; ideally want to run approximately 1 µg of RNA on the gel (should be about 1 µL of your sample)
Spray gel tray, comb, damns, and a small flask with RNAse Zap.
For small gels, melt 0.3 grams of RNA ONLY agarose in 30 ml 1X TBE (or 1X TAE) to the cleaned flask (for a 1% gel using Horizon gel boxes. If using something else, adjust accordingly).
Let the solution cool slightly, add 1 µL EtBr . Pour gel and let solidify.
For samples:
- RNA sample in a total of 8 µL of water (if RNA concentration is about 1 µg /ml , add 1 µL of RNA to 7 µL of RNAse-free water)
- 4 µL of sample buffer
- Mix samples
Sample Buffer (make FRESH!)
- 80 µL of concentrated dye (80% glycerol, 0.2% bromophenol blue, 0.2% xylene cyanol; this does NOT need to be made fresh!)
- 500 µL of formamide
- 120 µL of 37% formaldehyde
(1/10 of the recipe above is normally enough)
Load gel and include a 1 kb DNA ladder for size approximation; run at 100 volts about 45-60 minutes
Gel: should see 2 distinct bands corresponding to the 16s and 23s rRNA species. If RNA is intact, will not see smearing. Should also see lots of tRNA at the bottom.
Safety information
Formamide and formaldehyde are toxic and dangerous chemicals.
Handle with care in the hood!
Note
On occasion, I have run a mini-1X MOPS gel to validate RNA integrity especially if I do not have much sample left and really needed to use the RNA for an important experiment.