Intensity Loss for 200 nt Ladder Peak in the Agilent TapeStation RNA Assays

Created by Jasmin Manck-Goetzenberger over 1 year ago | Last modified by Geoff Pettitt 11 months ago

This Information Applies To: 2200/4150/4200 TapeStation systems. RNA ScreenTape and High Sensitivity RNA ScreenTape assays,TapeStation analysis software

Issue:

The 200 nt peak of the Agilent RNA ScreenTape and High Sensitivity RNA ScreenTape Ladder has lost its normal intensity. An example of this phenomenon is shown in Figure 1 with the actual 200 nt peak highlighted with a red circle:

Figure 1. RNA Ladder with rudimentary 200 nt peak in red circle

If the peak is below a certain intensity, the software will not assign it automatically. Instead, the next ladder peak, which is actually 500 nt in size, will be assigned incorrectly as the 200 nt peak (see Figure 1). You will also see an alert in the Sample Table indicating a missing peak (see Figure 2):

Figure 2. Sample Table alert indicating an issue with ladder peak detection

Resolution:

The 200 nt peak loses intensity, if:

Room temperature is too high, near the upper limit of 30 °C for the RNA ScreenTape assays (operating temperature for the assays: 14–30°C). In this case, run the assay again in lower temperature.
Heat-denaturation was not effective or not done at all. Heat-denature the ladder again according to instructions in the Quick Guide (72 °C for 3 min, place on ice immediately for 2 min, see also RNA ScreenTape Quick Guide, or High Sensitivity RNA ScreenTape Quick Guide). Ensure that the tubes used fit to your heat block or thermal cycler and that the heat block or thermal cycler is working.

If the 200 nt peak is not assigned in the software, it has no impact on quantification and RIN^e calculation as these parameters are calculated independently from the ladder. Quantification is based on the known concentration of the lower marker. The RIN^e algorithm evaluates the eukaryotic 18S or prokaryotic 16S rRNA signal against the fast region of the electropherogram (region between the 18S or 16S rRNA peak and the 5S region). However, if you have a rudimentary signal, you may assign a 200 nt peak manually. This change is needed, if you want to do DV₂₀₀ analysis and need to add regions correctly, or want to resolve the alert shown in Figure 2:

In the electropherogram or the gel image, right-click the peak or band area and select Add Peak (see Figure 3):

Figure 3. Right-click opens the electropherogram/gel context menu.

The software will automatically assign it as 200 nt peak and the alert will be gone. However, the Observations column in the sample table will show Sizing Changed (see Figure 4):