RIN Not Calculated or NA for Total RNA Samples in the Agilent Bioanalyzer 2100 Expert Software

Answer

This Information Applies To: 2100 Bioanalyzer, 2100 Expert software, RIN NA, Error 4501, RNA 6000 Nano kit (p/n 5067–1511), RNA 6000 Pico kit (p/n 5067–1513)

Issue:

The RIN is not displayed in the software or shows as NA. If the RIN is NA, the sample will be flagged in red and error 4501 will be present with a description such as Unexpected signal....

Steps to follow:

Check which assay was selected. A RIN, will only be calculated if a total RNA assay was selected. A RIN will not be calculated for the mRNA assays. If the wrong assay type was selected, prepare a new chip and rerun the samples selecting the appropriate total RNA assay.
Check that the lower marker and ribosomal peaks have been correctly assigned. To do this click Data in context menu and select the sample for which a RIN was not calculated. Then select the Peak Table tab. The peak designated as the lower marker has a black triangle above it. Turning the analysis on and off can help to determine if the correct peak was recognized as the lower marker (Figure 1). It is possible to assign a different peak as the lower marker by hovering the mouse at the top of the peak, right clicking and selecting Manually Set lower Marker. To check what peaks have been designated as the ribosomal peaks select the Fragment Table tab (Figure 2). If it looks like they have not been assigned correctly, please review page 67 of the Maintenance and Troubleshooting Guide for possible causes. For assistance checking the peak assignment, please contact you Agilent support representative.

Figure 1. How to check the lower marker assignment

Figure 2. How to check the assignment of the ribosomal subunits

Check if there is an Error tab present next to the Fragment Table tab (Figure 3). If this tab is available, click the Error tab to view the errors. If the Error tab is not there, the software hasn't flagged any errors for that particular sample. If Error 4501 is present, please continue reading.

Figure 3. Data context Error Tab

For the total RNA assays, the 2100 Expert software algorithm analyses the different regions of the electropherogram to calculate a RIN (Figure 4). The software also looks for anomalies: features that are not typical of the sample type. When a critical anomaly threshold is exceeded, a RIN will not be calculated and error 4501 will be present in the Errors tab. These anomaly thresholds prevent a RIN from being calculated when it might not be accurate. However, it may be possible to increase the anomaly thresholds to force the software to calculate a RIN. More information about the RIN can be found in this application note. Please note: RIN NA is a warning that the RIN may not be reliable for a particular sample (such as due to unusual noise/signals, ribosomal ratio, and other factors). Clearing the critical error message may yield a RIN, but Agilent does not guarantee the accuracy of this value. It is recommended to also perform a visual inspection of the data. In general, it is recommended to correct RIN errors only if the chip run is technically good and the unusual feature, for example an unexpected peak, is clearly distinguishable. The RIN value itself cannot be edited.

Figure 4. Electropherogram detailing the regions that are indicative of RNA quality.

The description next to the error should indicate which anomaly threshold needs to be increased. In the Setpoint explorer, click Local to modify a setpoint for the current sample or Global to modify a setpoint for all the samples on the chip. Then select Advanced from the drop-down list. In the RNA Integrity Number section, identify the anomaly threshold that corresponds to the error you wish to override. Increase the predefined anomaly threshold to one (Figure 5). One is the most relaxed value. The RIN will be now displayed in the Results tab, followed by a note: Anomaly Threshold(s) manually adapted.

Figure 5. How to edit the RIN anomaly thresholds