Discrepancy Between Agilent RIN and RINᵉ Values

Answer

This Information Applies To: 2100 Bioanalyzer, 4150/4200/2200 TapeStation

Issue:

Discrepancy between Agilent Bioanalyzer RNA integrity number (RIN) and Agilent TapeStation RNA integrity number equivalent (RIN^e) values

Resolution: 

First, please read the following application notes about RIN and RIN^e:   

• Performance of the RNA ScreenTape Assays on 2200 TapeStation System
• Performance of the RNA ScreenTape Assays on 4150 TapeStation System

As can be seen in the application notes, in general, we expect RIN and RIN^e to correlate well.

However, if differences in RIN and RIN^e are observed, we have the following recommendations:

1. Ensure that the sample preparation protocol is followed exactly as recommended for each assay. For example:

• For Bioanalyzer RNA assays, we recommend heat denaturing the samples before adding samples onto the chip. Although heat denaturation is optional to the workflow, it is highly recommended especially when comparing to RINe.  
• For TapeStation RNA assays, we recommend heat denaturing samples after adding sample buffer. 

Therefore, it is important to ensure the same samples heat denatured once for a Bioanalyzer run are not used for a TapeStation run. This will cause the samples to be heat denatured twice, negatively affecting the RIN^e value. Also, for heat denaturation, proper tools including but not limited to properly sealable consumables and PCR cyclers.  

2. Ensure that ScreenTape and Bioanalyzer assays of comparable sensitivity and specifications are used when comparing data across the two platforms.

3. Ensure samples are loaded according to the listed specifications for each Bioanalyzer kit and TapeStation ScreenTape assay, and only compare the results of the same samples.

Finally, although we expect the two to correlate well, RIN and RIN^e use different algorithms. RIN uses a proprietary neural network-based algorithm to assess the entire electrophoretic trace. For RIN^e calculation, the upper ribosomal peak is not taken into consideration. Correct assignment of the lower marker is essential for RINe calculation whereas ladder is not required. In contrast, RIN^e represents the relative ratio of the signal in the fast zone to the 18S peak signal. Also, given that Bioanalyzer and TapeStation are two different platforms, differences in electrophoretic patterns as well as in RIN values compared to RIN^e values may occur depending upon samples. In this case, setting a separate threshold or quality cut off for RIN and RIN^e is recommended. 

For Research use only 

PR7000-7979

For further information please refer to Agilent 2100 Bioanalyzer System Maintenance and Troubleshooting Guide Agilent Information Center > Agilent TapeStation Systems > ScreenTape Assays > Good Measurement Practices Agilent Information Centre > Agilent TapeStation Systems > ScreenTape Assays > Troubleshooting > Troubleshooting RNA Applications Agilent Information Centre > Agilent TapeStation Systems > ScreenTape Assays > Troubleshooting > Frequently Asked Questions