Degraded Bioanalyzer RNA Ladder or Samples

Answer

This Information Applies to: Agilent 2100 Bioanalyzer system, RNA 6000 Nano kit, RNA 6000 Pico kit, and Small RNA kit

Issue

The Bioanalyzer RNA Ladder and/or samples are degraded.

Background

Two examples of degraded RNA Ladders are shown in Figure 1. 

 

Figure 1. Electropherogram of a partially (left) and fully (right) degraded Bioanalyzer RNA Ladder.

Steps to Follow

RNA degradation can occur either before or during the chip preparation.

Degradation before the Bioanalyzer runs

The samples and ladder may have been degraded before loading onto the Bioanalyzer chip. To confirm if the ladder or samples were already degraded, consider checking their quality by another method such as with the Agilent TapeStation or Fragment Analyzer systems. 

Degradation during chip preparation

If the samples or ladder were not degraded before loading on the Bioanalyzer chip, then they may have been contaminated with RNase during the chip preparation or chip run. The source of RNase contamination must be removed. Perform the following steps before running a new chip:

• Follow the RNase decontamination process for the electrode cartridge as described in chapter 8 of the Agilent 2100 Bioanalyzer System Maintenance and Troubleshooting Guide.
• Use a new electrode cleaner chip (p/n 5065-9951). Two electrode cleaner chips are included with each box of RNA nanochips and three are included with each box of Pico and small RNA chips.
• Use a new box of RNase-free pipette tips.
• Use a new flask of RNase-free water.
• Use a fresh ladder aliquot.
• In case all ladder aliquots are suspected to be contaminated, reorder the ladder (Agilent RNA 6000 Nano ladder (p/n 5067-1529), RNA 6000 Pico ladder (p/n 5067-1535), Small RNA ladder (p/n 5067-1550)).
• Decontaminate lab bench with RNase ZAP™ or equivalent RNase remover.
• Decontaminate pipettes with RNase ZAP™ or equivalent RNase remover.
• Use a new unused reagent kit.

General precautions to avoid RNA degradation

• Wear gloves when working with RNA samples and reagents, and when handling accessories that will come in contact with the RNA sample.
• Use certified RNase-free plastics, disposable consumables, and water.
• Prepare ladder aliquots and store them at -70 °C.
• Use a dedicated electrode cartridge for RNA assays (p/n 5065-4413).
• Regularly clean the pin set and perform RNase decontamination of the electrode cartridge as shown in Table 1. Details are provided in chapter 8 of the Agilent 2100 Bioanalyzer System Maintenance and Troubleshooting Guide.

Table 1. Maintenance of the electrode cartridge

Learn how to effectively maintain and operate your Agilent 2100 Bioanalyzer system: 2100 Expert Software User's Guide on Agilent.com Agilent 2100 Bioanalyzer System Maintenance and Troubleshooting Guide on Agilent.com

For Research Use Only. Not for use in diagnostic procedures.

PR7001-3110