I guess the RIN went down during PolyA pulldown, which can happen. Did you use aliqouts or have there been freeze thaw cycles that also can reduce the RIN?

Two possibilities come to mind:

a) Your RNA got thawed during transport

b) Quality reduction happened during the handling at the sequencing contractor

Which company did you send it to (national or international)? And I suppose with -80 disaster you mean that you sent on dry ice? Can the company confirm that they received the box with enough dry ice left?

Hi Osama,

I agree with the previous answers from Torsten and Petra. It either comes from the PolyA pulldown, happened during transport, or at the sequencing platform. Could you make the following checks:

- as already suggested, did you send them on dry ice and can the company confirm that the samples were still frozen when they got them?

- did you make the polyA pulldown or did the company do it? In any case, is there enough sample left before/after pulldown? If positive, I would check those samples on a BioAnalyzer to determine whether it comes from that procedure.

- same for the transport: is there enough sample left before/after transport and before any other treatment at the platform? Again, comparing BioAnalyzer profiles for both could tell you whether something went wrong during transport (thawing essentially).

Ideally, prepare aliquots at every step of your procedure (at least 2µL, but 4 µL for example, in case you need to run it several times) so that you don't need to thaw a to-be-sequenced sample for the BioAnalyzer check up. Also, consider the electropherogram rather than the RIN itself, it might be more informative on the state of your sample.

Good luck,

Thank you all for the answers.

-The samples underwent one-two freeze thaw cycles after the initial RINs were read, one to make cDNA and one to send for sequencing (I am using Genewiz -USA). I tested RIN's on some samples after the first thaw cycles; all the reads were within +/- 0.5 points of the original reads. I do not think two freeze-thaw cycles actually can cause much deterioration.

-I used Fedex overnight service to ship the samples, and they delivered them within 20 hours, I had plenty of dry ice in the box. I will ask the company to confirm that the samples were on dry ice at receipt.

-I will confirm the profiles of the band profiles before and after shipping to see if there was really some degradation at some point in between.

-The company did not run the pull-down, they ran QC with Tapestation 4200 and Qubit. I guess that might have led to the discrepancy; since I used a Bioanalyzer 2100 nanochip. Could that be the issue?

I agree with Audery, I should have had multiple aliquots to avoid freeze-thaw cycles. I started doing that with all new extractions.

Thanks Again

Update: I sent the company my samples again - this time without thawing them at all. They ran the Tapestation study again, two samples that previously failed the QC - eRIN 5.5 and 5.0 passed the QC with eRIN of 6.2 5.9. So there was an element of freeze thaw in their. However, the remaining samples continued to have low eRIN and I had to replace them. The lesson is to aliquot RNA into multiple tubes (In multiple -80 freezers if possible), and expect differences between RIN (bioanalyzer) and eRIN (Tapestation). When I get the chance, I will re-run the samples on a bioanalyzer again.

Thank you for letting us know!

Hi All, an update, i had a new set of samples all of which had good RINs >8 using BioAnalazyer. I aliquoted them, and sent aliquots for RNAseq. The results of QC showed all samples to have lower RINs using Agilent Tapestation. One suggestion that original RINs were measured without denaturation. Insuppose Tapestation requires RNA denaturatuon.