RIN vs Volume?

[hbt]

I have a set of RNA samples prepared for RNAseq, however the RIN values and total volume on a few of the samples are less than ideal (<7.0 RIN and ~1.5ug total RNA) per sample

I have now produced a second set of samples (direct biological repeat) with much better RIN values (~8.6) but still low volume of total RNA.

My question is whether I would get a better set of results by combining the two samples, thereby reducing the RIN value to and average of the two samples but providing the sequencing centre with a much greater volume of RNA to library prep from? (and presumable reducing the number of duplicates). Or do I just have the second set of samples sequenced as they are the best quality samples I have available?

In other words, which has a greater affect on the quality, coverage level and level of duplicates of the dataset? RIN value or total volume?

The first set of samples also had some poor 260/230 ratio values, does this have much affect on the RNAseq chemistry and could it compromise the quality of the results further?

The samples are to sequenced (single end 50bp) on 1/3 of an illumina Hiseq2000 lane each, following RiboMinus treatment

any thoughts would be greatly appreciated

Thank you

[pmiguel]

Originally Posted by hbt

The first set of samples also had some poor 260/230 ratio values, does this have much affect on the RNAseq chemistry and could it compromise the quality of the results further?

The samples are to sequenced (single end 50bp) on 1/3 of an illumina Hiseq2000 lane each, following RiboMinus treatment

any thoughts would be greatly appreciated

Thank you

Are you constructing the libraries?

RiboMinus depletes degraded rRNA poorly. Why not use RiboZero?
As long as you have a good insertion point into your protocol, then slightly degraded RNA will work fine if you RiboZero deplete. But you don't want to do a polyA+ purification as well.

260/230 is a measurement of the relative absorbance of your DNA (260) against other stuff capable of absorbing at 230 nm. That would include acetic acid or salts of acetate or the shoulder from a guanidinium salt. Other than the effect of the salt itself on hybridization of ribo depletion oligos to your sample rRNA, neither of these should have much effect on this step. After ribo depletion, you will desalt your sample anyway. (Use a Zymo column, not ethanol precipitation or that wretched Invitrogen "concentration" device.)

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Phillip