Is the a way to check the quality of nuclear RNA on agarose gel? Since there will not be any rRNA there, I was not sure if it can be run on a gel or the only way to assess the quality if by Bioanalyzer. Thank you!
If you have a lot of RNA which you won't care wasting in electrophoresis, it can be easily done. However, how good the gel appear, it can't be said, as RNA gel without ribosomal RNA would be just a smear. A major share of extracted RNA is rRNA and its removal significantly reduce the RNA amount and concentration in sample. This is the reason for bioanalyzer/tapestation analyses which are ofcourse more accurate/sensitive and use just a very small amount of sample.
As an in between for gel or bioanalyzer, you can use the Qubit RNA IQ assay, which gives a score out of 10 of how much large or structured RNA is in the sample compared to small RNA
Abhijeet Singh But without rRNA, there will not be any distinct bands on the gel. So is it actually possible to comment on the quality of RNA by running it on the gel? Approx how much RNA should be loaded for it to be detected on the gel? I will be using bioanalyzer for my real experiments. Currently I need an easy technique that I can do in my own lab quickly (We do not have or own bioanalyzer yet)
Shaun Clare Ya Qubit is good as well. But I feel gels give better info on if the RNA is intact or degraded.
The Qubit RNA IQ assay uses two dyes, one that binds to structured or large RNA (rRNA, mRNA and tRNA) and another dye that binds to small or degraded RNA. Plus you'd only be using 1ul and is also the fastest since you can run a sample in a couple of minutes. I think all methods are appropriate but it depends on how much time and money you have to spare
Thanks for this detail Shaun Clare . I would use qubit and bioanalyser when I do my real scRNA seq /snRNA seq experiments but I wanted a less expensive and handy techniques while I am optimizing a protocol. So I was wondering if agarose gel would help.
Yes, you can do a quality check on a gel (tentatively) for mRNA. An mRNA smear in the gel can be analyzed based on the intensity of the smear where you expect the average linear size/length of the expressed fraction. For example, in a pea genome, the avg length of an expressed fraction is ~2.8k, so while running an mRNA sample I would look for an enriched intensity at 1.5-3.5 k reference bands/ RNA-ladder. Also, check that the sample should not have too much intensity accumulation after 100 bp. Of course, this crude method won't work for snRNA.
However, I would always prefer to run a bioanalyzer, if a downstream application is expensive and experimental success largely depends on the sample quality.
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