What testing should be performed to assess the quality of an RNA sample?

All RNA samples should be assessed spectrophotometrically (diluted in 10mM Tris, pH 8.0), and electrophoretically, and should meet the following specifications:

  • Total RNA concentration by A260 should be greater than 40 µg/ml
  • A260: A280 ratio should be 1.8 to 2.0
  • A260: A230 ratio should be greater than 1.7
  • Analysis of ~100ng of total RNA on an Agilent Bioanalyzer using an RNA 6000 Nano LabChip, or analysis of 1.5 μg of total RNA on a denaturing 2.0% agarose gel containing ethidium bromide (0.5 μg/ml) should contain sharp 28S and 18S rRNA bands, with no smearing at their low molecular weight edge. The 28S:18S band intensity ratio should be ~2:1. When utilizing the RNA 6000 Nano LabChip for RNA analysis, the RNA should have a RIN (RNA integrity) score of 7.0 or higher.

In addition to the above quality control tests, the RT2 RNA QC PCR Array for human (PAHS-999), mouse (PAMM-999), or rat (PARN-999) can be used. These arrays allow the rapid assessment of high and low housekeeping gene expression levels, reverse transcription and polymerase chain reaction efficiency, and genomic and general DNA contamination.

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