I hate having to run MOPS PFA agarose gels with DEPC and everything just to verify my RNA integrity. I came across an alternative protocol that uses ordinary household bleach in the gel to inhibit RNases and was wondering if anyone here has tried it and what the success was like? Or any helpful insight is welcomed also.

here's a protocol I put together based on what was done in that paper.

For a 50ml gel IN a RNase-free 250ml Erlenmeyer flask Mix: 5 mls of 10x TAE that I gave you 42.5mls ultra purified water 0.5g of the agarose I gave you 2.5mls of household bleach (6% sodium hypochlorite, use clorox)

Incubate 5-10 minutes.

Microwave for the minim amount of time on the lowest heat setting that’s needed to completely solubilize the agarose, you don’t want evaporation adjusting your solutions volume Check your volume with a sterile 50ml serological pipette, if you have lost more than 5% volume I would redo, using less heat.

Add 3µl 10mg/ml ethidium bromide, swirl to mix. Pour in casting frame, allow to cool, submerge in 1xTAE

Combine 1µg of your total RNA, 2µg RNase free 6x loading dye and q.s to 12µl in RNAse feree water, you can use the water from the qiagen kit for this. Run at 100V, about 30 min, get good scanned image of it and use Photoshop to assess the band intensity. The 28S should be twice the brightness of the 18S in a sample with little to no fragmentation.

  • 1
    $\begingroup$ I also hated the denaturing agarose RNA gels, but I switched to native gels using Faster Better Media's Lithium Boric Acid buffer at 0.5X concentration, my gels look much better, though I get 2 bands in each lane because it's not denatured. How are you using the gels to check for RNA integrity? If I see a band at the right size I've been calling it good enough and moving on. How precise does a gel need to be? $\endgroup$ – user137 Sep 3 '14 at 21:42
  • $\begingroup$ Looking at the small and large subunits. The large one should be 2x as bright in an unfragmented sample of total RNA. The gel needs to not exhibit any exonuclease activity, not much more than that needed $\endgroup$ – rhill45 Sep 3 '14 at 21:53
  • $\begingroup$ Maybe that's why I've been able to get away with my gels, I'm making RNA through In Vitro Transcription, you must be isolating it from cells. $\endgroup$ – user137 Sep 3 '14 at 21:57
  • 1
    $\begingroup$ What is your ultimate objective? What do you need RNA for? The rigor of the test depends on that. For usual RT-PCR I heat RNA with the 2x loading buffer that contains formamide and run it in normal agarose gel with TAE/TBE at 120V. $\endgroup$ – WYSIWYG Sep 4 '14 at 5:58
  • 1
    $\begingroup$ Why not just run the RNA on an Agilent Bioanalyzer nanofluidics machine (or something similar)? It's fairly cheap and quick, and I'm sure a lot of core services offer it now. Of course, I am making certain assumptions about funding and technology availability here... $\endgroup$ – Cantona's Collar Feb 27 '15 at 1:19

I don't think bleach can denature RNA. Bleach is an oxidizing agent and it will damage the RNA. Also, the protocol mentions addition of hypochlorite before heating which I think is illogical because heat will decompose it.

For testing RNA integrity you need not make a denaturing gel. What you can instead do is to heat the RNA with the 2xRNA loading buffer that contains 95% formamide and run it in a normal TBE/TAE agarose gel. Heating with formamide denatures the RNA permanently.

Denaturing gels are only essential for Northern Blots.

You can also run your sample in a bioanalyzer to check for RNA integrity and this is always recommended for RNAseq experiments (even for microarrays but I cannot comment much on that because I have never done it and am not aware of the usual practice).

  • $\begingroup$ I'm telling you I didn't believe it either but it works quite well. Don't add it to the agarose before microwaving and use 6%. Also the rna will be denatured already from most any purification procedure especially the standard ones that use guanidine. works good for a teaching lab (cheap and easier) and I think this protocol focus's on RNAse removal or inactivation. +1 for the heating recc but I'm weary to heat rna unless it can be flash heated. I may Ty this next time however. $\endgroup$ – rhill45 Feb 27 '15 at 2:16

The bleach is not to denature the RNA but to destroy and RNase that might be lurking in your agarose or buffer - heat doesn't kill RNase easily so the boiling doesn't help. Then just run a normal agarose gel (TAE/TBE or borate) and look at the relative intensities of the LSU and SSU RNA. The bleach gels work very well for me and I've got very good RNA-Seq results out of RNA analysed this way (I don't have access to a bioanalyser).

  • 1
    $\begingroup$ Please add some references to your answer. $\endgroup$ – another 'Homo sapien' Jan 26 '17 at 13:38

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.