total RNA quality for library construction

[neveaire]

Hi Everyone -

I've been given some RNA that was extracted from snap frozen mouse muscle (leg). The bioanalyzer tells me that the RNA is degraded (RIN ~2.5-3) with fragment sizes centered around 200-500 bases. Is it possible to make a library out of this? Ideally I'd like to ribodeplete the sample first.

Any thoughts appreciated.

[mnkyboy]

It is possible if you follow a standard Illumina RNA prep then DSN treat that may help you with your degraded sample (also well help with ribosomal).

This is a method that works very well for FFPE samples and may work with degraded RNA.

[neveaire]

Can you explain why DSN might help with degraded RNA? I understand the steps involved but not the reasoning.

It is possible if you follow a standard Illumina RNA prep then DSN treat that may help you with your degraded sample (also well help with ribosomal).

This is a method that works very well for FFPE samples and may work with degraded RNA.

[mnkyboy]

I think it will remove most of your abundant products of RNA degradation allowing the lower mass amount of intact RNA to be detected. This was something Illumina claims and I have not tested it yet. They say DSN works for all low quality RNA samples including FFPE.

[pmiguel]

If you denature a DNA sample and then allow renaturation for a limited amount of time, only those fragments of DNA that are most highly reiterated (high copy) in a sample will have time to form double stranded DNA. Why? Just because high copy fragments in a solution have a better chance of colliding with a complement than low copy--it takes them less time to do so.

Ribo depletion methods generally rely on the ribosomal RNA being intact because they bind a just a few sites on the large and small ribosomal sub-unit RNAs. If you had biotinylated oligos that bound every segment of the ribosomal RNA, then you could remove it all no matter how degraded it was.

A DSN, "Double Stranded Nuclease" will degrade the double stranded stuff, leaving lower copy DNA behind.

In just about any total RNA sample >85% of the RNA will be ribosomal RNA. So even if you completely skip ribo depletion, DSN normalization will degrade most of the rRNA cDNA before anything else.

The down-side is that once you normalize, you no longer can obtain quantitative data from your RNA-seq sample because you have heavily biased downward the higher copy RNA counts. But for de novo transcriptome work, it could work fine.


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Phillip