RIN for a sample is computed using several characteristics of an RNA electropherogram trace, with the first two listed below being most significant. RIN assigns an electropherogram a value of 1 to 10, with 10 being the least degraded. All the following descriptions apply to mammalian RNA because RNAs in other species have different rRNA sizes:
The total RNA ratio is calculated by taking the ratio of the area under the 18S and 28S rRNA peaks to the total area under the graph, a large number here is desired, indicating much of the rRNA is still at these sizes and thus little to no degradation has occurred. An ideal ratio can be seen in figure 1, where almost all of the RNA is in the 18S and 28S RNA peaks.
For the height of 28S peak, a large value is desired. 28S, the most prominent rRNA species, is used in RIN calculation as it is typically degraded more quickly than 18S rRNA, and so measuring its peak height allows for detection of the early stages of degradation. Again, this is seen in figure 1, where the 28S peak is the largest, and so this is good.
The fast region is the area between the 18S and 5S rRNA peaks on an electropherogram. Initially, as the fast area ratio value increases, it indicates degradation of the 18S and 28S rRNA to an intermediate size, though the ratio subsequently decreases as RNA degrades further, to even smaller sizes. Thus, a low value doesn't necessarily indicate either good or bad RNA integrity.
A small marker height is desired, indicating only small amounts of RNA have been degraded and proceeded to the smallest lengths, indicated by the short marker. If a large number is found here, that indicates that large amounts of the rRNAs have been degraded to small pieces that would be found closer to this marker. This situation can be seen in the poor quality RNA electropherogram found in figure 2, where the height of the peak over the marker (far left) is very large, so the RNA has been greatly degraded.
In prokaryotic samples, the algorithm is somewhat different, but the Agilent 2100 Bioanalyzer Expert software is able to calculate RIN for prokaryotic samples now as well. The difference likely arises from the fact that, while mammalian samples have 28S and 18S ribosomal RNAs as their predominant species, prokaryotic RNAs have the sizes shifted slightly smaller, to 23S and 16S, so the algorithm must be shifted to accommodate that. Another crucial fact about calculating prokaryotic RNA integrity numbers is that RIN has not been validated to the extent that it has for eukaryotic RNA. It has been shown that higher RIN values correlate with better downstream results in eukaryotes, but this hasn't been done as extensively for prokaryotes, so it may mean less in prokaryotes.
These electropherograms for calculating RIN are done using the Agilent Bioanalyzer machine, which is capable of performing electrophoresis and generating the electropherograms. The Agilent 2100 software is uniquely able to perform the RIN software, as the exact algorithm is proprietary, so there are additional important RNA electropherogram features that are used in its calculation that are not publicly available.