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Next Generation Sequencing: RIN Analysis

RIN stands for RNA Integrity Number, and is essentially a metric of the quality of RNA, scaled from 0-10, based upon the relative size and ratio of the 18S and 28S peaks in a given RNA sample. If the RNA is more degraded, one or both of those peaks will be smaller than expected and the background noise will be higher, resulting in a lower RIN score.

RIN of 8.9

This graph shows a Tapestation trace of an RNA sample with a RIN of 8.9. The 18S and 28S peaks are in the expected locations and are relatively narrow, with limited signal between peaks and extending down towards the lower marker.

RIN of 7.4

The graph above shows a Tapestation trace of an RNA sample with a RIN of 7.4. Notice that the 18S and 28S peaks are still crisp and well-defined, but that the amount of background signal between the peaks and at lengths shorter than 18S has increased compared to the first trace.

RIN of 4.6

This sample had a RIN of only 4.6. The 18S and 28S peaks are significantly lower and broader, and much more signal can be seen towards the left end of the graph. This is indicative of more substantial RNA degradation.

In our core, we recommend using the SPIA RNA kit for sequencing if your samples have RINs of less than 6.0, to avoid sequencing bias from potential mRNA degradation. Samples with RINs above 6.0 are typically high enough quality for poly(A) priming for mRNA isolation to accurately represent the transcripts present.