How to test RNA quality

In labs where realtime PCR is being used to evaluate gene expression, it is crucial to test the quality of extracted RNA before proceeding with reverse transcription (RT). As a minimum, a portion of the RNA sample should be run on a gel. You should expect to see 2 strong bands, the 18S and 28S RNA. If you see a smear then you can forget about continuing the experiment with that sample. While it is nice to have an idea of the RNA quantity by checking the OD 260nm reading on a spectrophotometer or a nanodrop, this is NOT sufficient as it in no way gives you an indication of the quality of the RNA.

One of the problems with running RNA on a gel to assess quality is that it uses up several ul of your precious sample. It can also be rather difficult to judge by eye whether the sample has started to degrade or not. A better way of assessing RNA quality is to run a portion of your sample (1ul) on an automated capillary electrophoresis system.

There are currently 2 systems on the market that will do this. The Experion and the Bioanalyzer. There is some debate over which system gives better data but the reality is that they are both comparable. The Experion system has the advantage in being far easier to use since it uses an automated priming station to fill the test chip rather than a manual syringe like the Bioanalyzer. The test chips used in both systems are made by the same manufacturer (Caliper Life Sciences) but they are not interchangeable. The Experion test chips have a longer shelf life, so that might be important to some labs.

Here are some examples of data collected using realtime PCR and the effect of degraded RNA.

Assessing RNA Quality and Quantity in siRNA Gene Silencing Experiments

Effect of degraded RNA on qPCR Data Quality

Degraded vs Intact RNA 

For more information on the Experion download the Experion Brochure.

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