Help with determining RNA quality for RNA SEQ

[lior lobel]

Hi All,

I'm new to RNA-Seq, so I'm not that familier with RNA quality checks for RNA seq. I have 6 total bacterial RNA samples, that I want to carry for rRNA depletion using the RiboZero kit. Before doing that I ran the samples on agarose gel and got a strange result (please see attached). It seems that all the samples have a huge band around 100bp.. what this means? is that degraded RNA? or maybe DNaseI degraded DNA leftovers?
Any help interpreting this gel will be highly appreciated!

Thanks and best,

Lior

[HelenaSC]

Hi Lior,

Don't you have the possibility to check your samples with a Bioanalyzer or similar??
I think it would be better...

[lior lobel]

Hi Helena,

Just yesterday did the bioanalyzer analysis. please see attached. This is after rRNA depletion by RiboZero and by mistake the analysis was done with Eukaryotic reference.

The big 100-140 nt peak is tRNAs and sRNAs, right?

Thanks and Good weekend,

Lior

[HelenaSC]

Yes, you are right, the peak should be miRNA, tRNA and so on.
Good weekend!

[mRNA123]

This paper has a nice method of assessing RNA quality not based on rRNA. Essentially they look for degradation from the 3' end based on RT-qPCR products from the same RNA using the same forward primer and various reverse primers. Assuming 3'-to-5' decay, a sample with extensive RNA decay will have less of the RT-qPCR products near the 3' end.

http://www.gene-quantification.de/bjoerkmann-et-al-delta-amp-bdq-2016.pdf