RIN and 28s/18s ratio
Getting back into the RNA biz (after way too many years) with the aim of RNA seq...
I am running some trials to determine the best way to produce the high quality RNA from a cultured cell preparation as determined by coverage after sequencing.
I am assessing my first RNA preparations before moving onto cDNA and subsequent library production.
Perhaps I'm being over-cautious, but can someone please explain the significance of both the RIN and 28s/18s ratio.
The trial looks very good as far as these numbers are concerned as determined by a bioanalyzer profile from a RNA 6000 nano assay.
Here are the details:
I prepared total RNA via four methods; 5 million cells per preparation, 4 sets of duplicates, 8 samples total.
Concentration, as determined by nanodrop, ranged from 242 - 429 ng/ul.
The 260/280 ratios range from 1.94 - 2.04; the 260/230 ratios range from 1.99 - 2.23.
The samples were diluted to 50ng/ul for the bioanalyzer run.
As per the Agilent instructions, the dilutions were heated at 70C for 2 minutes prior to being loaded onto the chip.
The profiles from the bioanalyzer show precise 18s and 28s peaks in all but one sample.
In that sample the 28s peak is somewhat more broad than the other samples.
The bioanalyzer software has returned the following information:
The concentration in the dilutions (as determined by the bioanalyzer) range from 40-57 ng/ul.
7 samples have a RIN of 10; the last RIN (the sample with the broader 28s peak) is 9.9.
The 28s/18s ratios range from 2.1 - 2.4.
I also ran a few of these samples at 25ng/ul as I wasn't sure what would be appropriate.
These appear to correspond with their higher concentration counterparts.
Should I run higher RNA concentrations now or in the future?
Should I be hoping for 28s/18s ratio's of closer to 2.7?
How reliable is the RIN as an indicator of quality?
In truth, I am very encouraged and will continue to process these samples into sequencing libraries.
I'm just looking for any feedback on how to make quality assessments, i.e., what are the standards of excellence, what are the acceptable parameters, etc.
thanks in advance