The RNA Integrity Number (RIN)

Although the 28S/18S rRNA ratio is a reasonable way to estimate RNA integrity, it is
not ideal and in particular in the context of the Bioanalyzer, which does not run under
denaturing conditions sometimes the ratios do not reflect the exact level of RNA
degradation. Besides the ratio of the 18S to 28S ribosomal RNAs, Agilent has
introduced a software algorithm that takes the entire electrophoretic trace into
account to help scientists estimate the integrity of total RNA samples.

The RNA Integrity Number (RIN) software algorithm allows the classification of total
RNA, based on a numbering system from 1 to 10, with 1 being the most degraded
and 10 being the most intact. In this way, interpretation of an electropherogram is
facilitated, comparison of samples is enabled which ensures better reproducibility.

Although the RIN tremendously facilitates the assessment of the integrity of RNA
preparations and makes it much easier to compare samples or RNA isolation
procedures, it is not possible yet to determine ahead of time whether an experiment
will work or not. For example, a RIN of 5 might not work for a microarray experiment,
but might
work for an RT-PCR experiment. In general, RINs higher that 7-8 seem to be working
well in most of our experiments. RINs below 7 require extra validation studies before
we are able to conclude “how bad is still good enough".

It is important to realize that we assume that the integrity of the rRNA species
provides a good indication for the integrity of the mRNA species, which is most of the
time the RNA species that we are interested in. Although this is a reasonable
assumption, it is by no means a fact. As rRNA transcripts should be considered
stable (half-life of 4-5 days), a significant fraction of the mRNA could be completely
degraded while there still could be enough high molecular weight rRNA left.

The suitability of the RNA sample is strongly dependent on the source of the RNA and
the application intended for it be it a qualitative or quantitative application. If for
example one would like to determine the presence of a particular exon within an
mRNA isoform, it would not matter if the total RNA would have a RIN of 1. As long as
the splice variant can be detected either by size or by sequencing the PCR fragment
obtained form the sample. If one would like to perform RT-PCR to show the presence
of a particular transcript within a cell type or tissue, again the RIN could be between 1-
5 and still one would be able to conclude that the transcript is expressed in that
particular tissue. One should be careful by concluding that the transcript was absent
if the RT-PCR gave negative results and was performed on RNA with a RIN below 5,
specially if the transcript has a short half-life. For every type of quantitative analysis,
performing experiments with RNA with a RIN below 6 should be done with caution.
Specially, if one would be using freshly harvested cultured cells or freshly harvested
tissue from a laboratory animal, the RIN should be definitely higher that 7.5-8
otherwise there was a problem with the way the RNA extraction procedure was
performed. In some situations one has no choice, as when a sample has been
stored for many years in the freezer and/or if the tissue has been fixed with
formaldehyde. Isolation of RNA from these type of tissues can not result in perfect
quality RNA and every one should decide for themselves if they will use the sample
and for what purpose. Needless to say that for highly demanding (and expensive)
assays like microarray analyses, one would like to have RIN values higher that 7 for
frozen tissues, but definitely higher that 8 for tissue cultured cells.

Further reading:
Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S,
Menzel W, Granzow M, Ragg T.
The RIN: an RNA integrity number for assigning integrity values to RNA
BMC Mol Biol. 2006 Jan 31;7:3.

Next: RNA quality assessment using the Bioanalyzer, The Good, the Bad and the Ugly
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