polyA library preparation and RIN values

[renuka22]

Hi there,

I am new to this forum and had some questions about the RNA bioanayzer data and nanodrop readings. I got a good concentration of 200-300ng/ul when I analysed my samples by nanodrop whereas on the bioanalyzer, the compay was not able to obtain any RIN value (Nobands obtained, not even a smear, only some clear area seen on the gel). They concluded saying all the RNA is degraded. Also, they say they are not able to make polyA library as the concentration after polyA shows only around 0.1-0.2 ng/ul and it is very low to get any sequencing results. Anyone can suggest something regarding this, coming from a totally inexperienced person..

Thanks

[nucacidhunter]

Nanodrop QC is only good to detect contaminants not for quantification. RNA quantification can be done using Qubit or similar method and integrity by gel or Bioanalyzer-like instruments. Quantification by Bioanalyzer is Ok for most application though it is not very accurate. I tend to agree with your service provider.

[pmiguel]

Has anyone bought a "Nanodrop One"? It is supposed to be able to analyze your spectrum of your sample and deconvolute the spectra of nucleic acids, protein, guanidine and phenol. That is, supposedly it can correct for the presence of the latter three common spectral contaminants of nucleic acid preps/methods and give you an accurate nucleic acid concentration.

I don't imagine it will be able to distinguish between RNA and DNA, so genomic DNA prep concentrations will still usually be wrong. But for RNA preps, DNA usually isn't a major contaminant.

If it works and renuka22 had one, it would have saved him or her some troubles...

--
Phillip

[renuka22]

No, I have never used this. Would have been a good idea though..

[renuka22]

Does doing polyA treatment lowers RIN values? My polysomal RNA values showed good RIN values of 9.5..but when I did the polyA, it was in the range of 5-6. The concentration could not be detected by qubit high sensitivity kit. Is the polyA treatment degrading the samples?

Thanks

[nucacidhunter]

RIN number is valid only for total RNA and one of the main factors in its calculation is 18S/28S ratio. With a successful polyA selection rRNA would be excluded so RIN number is not meaningful. If after polyA selection you still see 18S or 28S bands then it has been less successful. See Agilent application note about RIN number: https://www.agilent.com/cs/library/a...989-1165EN.pdf