Recently, I am working with a newly isolated yeast RNA for RNA sequencing. I have sent my RNA samples for RIN analysis using Agilent Bioanalyzer. However, I have received error results for all 12 samples. Based on the results attached, the 18s/28s ratio and RIN cannot be calculated. The retention time for 18s/28s for all samples were not within the start time/end time mentioned in the report. Is that why the RNA ratio and RIN cannot be calculated? What are your suggestion/troubleshooting for these results. Is the manually adapted results accurate? Can I use this results as reference for RNA sequencing
Hi
receiving errors from the bioanalyzer means a lack of quantity or quality (RIN
hi Frederic,
Thanks for your reply.
But based on the peaks for 18s/28s RNA generated by bioanalyzer for each samples looks good though. There are distinct peak for 18s and 28s as per attached in the attachement but the time that the peak came out is not the same time as start time/end time stated at the bottom of the report. Kindly refer to this attachment.
Btw, i did run the RNA samples on electrophoresis gel prior to bioanalyzer and the results did look good.
Hi,
a feedback from one of our engineer is that you have really good samples, maybe your RINs will reach between 9 and 10, at the only condition to dilute your samples by 5. in fact your samples are too concentrated, the maximum they use are under 500ng/ul, impossible for the software to distinguish the size marker and therefore to assign right values for RIN and positions. maybe you'll need to reassign the pics for the size markers.
fred
Looks like the samples themselves are OK, but retention times across wells are inconsistent. The most common reasons for such an issue are the gel (freshly prepared? correctly centrifuged?), priming (setting of the priming station? duration of priming?) and sample concentration (very high in your case; try diluting next time). Have you checked the lower marker peaks for each lane? If these don't appear at the correct time/position, sample peaks will also be off. Before proceeding to RNA-Seq, I'd definitely dilute these samples and run another chip using fresh reagents and paying extra attention preparing the chip - good luck!
Thanks Fred & Dominik for your feedbacks & suggestions. Really glad to be reading them. I can't answer those questions because we outsourced the samples for bioanalyzer analysis. But i will try to discuss with the company.
Thanks again!
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