I confirm that RIN values above 7 are quite good for clinical samples and cannot alway be obtained from such samples.

Morevoer, the crucial point is the homogeneity of RIN values between several RNAs that you aim to compare in a transcriptomic analysis : gene expression measured from a sample with RIN=7 and another with RIN=9 for instance, might be biased, only based on the differential quality level of total RNA (apparent lower expression of a given gene in RIN7 sample vs RIN9 sample).

This doesn't mean that you should systematically eliminate from your study the samples with "extreme" RIN values, especially if you work on precious and rare samples : just go on with all samples till the end of your transcriptomic analysis (especially RT-qPCR), but keep in mind the initial heterogeneity of RNA quality when interpreting the results, and check any relationship between lower RIN value and lower apparent expression (this is facilitated when comparing groups of individuals).

If you perform a RT-qPCR quantification on one or a few genes, such discrepancies could have no effect on the result, depending on the genes sudied (sensitivity of their RNA products to ribonucleases). Indeed, the RIN score mainly relies on ribosomal RNAs integrity and thus provides an indirect evaluation of the integrity of other RNA species (the one you quantitate).

However, if you wish to analyze many genes (NGS, microarrays, qPCR arrays...), the RIN scores of all samples studied must be very close (ideally about 0.5 RIN unit between the highest and the lowest RIN value ), to garantee reliable results for all genes.

Hi Aniruddha, RNA with RIN number 7.1 is acceptable and can be taken for further transcriptome study. It is always difficult to get RNA with RIN > 9 from clinical samples but not impossible. I regularly work on these clinical samples. For microarray with 3' biased probe you can absolutely go ahead. For microarray with probes which spans the whole length of transcript, there might be little concern as some times you get fragmented RNA and you might loose the 5' end fragments of your mRNA . For RNA-Seq study people will ask for a little high RIN vaue RNA but they might accept the sample. I think you can go ahead with your transcriptome study with this RNA sample. Best of luck.

Thanks for you feedback. I am planning to do RNA-seq. They are treated with DNAase twice and before processing for RNA extraction I stained and checked if there are dead cells, there wasnt any. So, I think this is the best I could get. Also, I guess the quality from these isolation and traces might also depend on the cell type rite ? I mean Neutrophils are quite active cells gives the first line of defence and also they have lots of RNAase in them too.

Hi Aniruddha, I will suggest you to try once with Trizol after isolating the Neturophil from whole PBMCs. Just wash twice with Phosphate Buffer Saline (Physiological pH) and proceed with the isolation following the Trizol protocol. I don't know if you have samples to try it again. I also don't know how you isolated previously. May you have used KIT to isolate the RNA. Have you checked the RNA RIN number before treating with DNAse because many reports suggested that DNAses carry small amount of RNAes as a contamination and has been found that the RIN number decreases after the DNAse treatment but how much I don't have the idea.

Hi Aniruddha, that RIN is actually not too bad for alot of clinical samples. If you're absolutely satisfied that you cannot get better RNA I would go ahead with the RNA-seq.

For sub-optimal RIN values many service providers and institutions recommend using the EpicentreBio Ribozero rRNA depletion kit in conjunction with the script-seq library preparation methods. Supposedly this method of library preparation is less sensitive to degraded input RNA due to the variety of rRNA loci that the Ribozero kit targets.

I am in the process of preparing an experiment with clinically derived RNA which is moderately degraded. I am opting for these same methods, I'll let you know how I get on.

Best of luck :)

RIN of 7.1 should be perfectly fine. If you have any doubt on the integrity and continuing on, use the Ovation RNA-Seq System V2 from NuGEN to make the library preps. This system works wonderfully with great quantities and quality to work with going into sequencing

Thanks.. this discussion is already very helpful. I have attached the bioanalyser tarce here for the sample with the question.

It looks pretty degraded based off of the ratio of 28s and 18s

RIN of 7.1 is ok. The trace does look a bit of disappointing - you do have degradation. If this is the best you can get out of the method you are using or if this is a precious sample, I will definitely go ahead with it.

I confirm that RIN values above 7 are quite good for clinical samples and cannot alway be obtained from such samples.

Morevoer, the crucial point is the homogeneity of RIN values between several RNAs that you aim to compare in a transcriptomic analysis : gene expression measured from a sample with RIN=7 and another with RIN=9 for instance, might be biased, only based on the differential quality level of total RNA (apparent lower expression of a given gene in RIN7 sample vs RIN9 sample).

This doesn't mean that you should systematically eliminate from your study the samples with "extreme" RIN values, especially if you work on precious and rare samples : just go on with all samples till the end of your transcriptomic analysis (especially RT-qPCR), but keep in mind the initial heterogeneity of RNA quality when interpreting the results, and check any relationship between lower RIN value and lower apparent expression (this is facilitated when comparing groups of individuals).

If you perform a RT-qPCR quantification on one or a few genes, such discrepancies could have no effect on the result, depending on the genes sudied (sensitivity of their RNA products to ribonucleases). Indeed, the RIN score mainly relies on ribosomal RNAs integrity and thus provides an indirect evaluation of the integrity of other RNA species (the one you quantitate).

However, if you wish to analyze many genes (NGS, microarrays, qPCR arrays...), the RIN scores of all samples studied must be very close (ideally about 0.5 RIN unit between the highest and the lowest RIN value ), to garantee reliable results for all genes.