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Question 13 of 20 Intermediate Interpretation

What is an RNA integrity number, and how should it change what you do?

Short answer

The RNA integrity number, or RIN, is a score from 1 to 10 computed from a capillary electrophoresis trace of a whole RNA sample, where 10 is intact and 1 is fully degraded. A low value warns that expression measurements will be biased, so you either repeat the extraction or choose methods that tolerate fragmented RNA.

Why

The older measure was the ratio of the 28S to 18S ribosomal RNA peaks, with about 2.0 counting as good. Schroeder and colleagues showed that this ratio is unreliable and built the RIN instead, using several features of the whole electropherogram rather than two peaks. Their data showed apparent expression of housekeeping genes by real-time PCR tracked the RIN closely, while the ribosomal ratio correlated only weakly.

Degradation does not hit every transcript equally, so a comparison between a RIN 9 sample and a RIN 4 sample can produce differences that are entirely technical. The practical responses are to repeat the extraction from better-preserved material, to match RIN across groups so that degradation is not confounded with treatment, or to switch to a method built for fragmented RNA. Short amplicons help in qPCR, and primers placed toward the 3' end help when poly-A selection is used.

Formalin-fixed tissue is almost always fragmented, so library kits for that material use ribosomal depletion and random priming. A RIN threshold has to be chosen for the application rather than copied, and it should be recorded for every sample.

A strong answer covers

  • Score from 1 to 10 from the whole electropherogram
  • More reliable than the 28S to 18S ratio
  • Degradation biases apparent expression
  • Match integrity across groups or repeat the extraction
  • Short amplicons and specialized kits for fragmented RNA

Common mistakes

  • Comparing groups whose RNA integrity differs systematically
  • Treating the 28S to 18S ratio as equivalent to a RIN
  • Applying one RIN cutoff to every application regardless of method