RNA Quality

Impurities in RNA samples have an adverse effect on both the labeling efficiency and the stability of the fluorescent labels that are used. Therefore, the success of your microarray experiment depends on the quality of your prepared RNA. It is important to check the quality of the RNA after each thawing and before starting the labeling reaction, since impure RNA samples might degrade during the thawing process.

RNA samples must be free of contaminating proteins and other cellular material, organic solvents such as phenol or ethanol, and salts. One measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm. The A260:A280 ratio for RNA samples of acceptable purity should be between 1.8 and 2.

RNA samples should also show no signs of degradation. A high-quality preparation of mammalian total RNA is characterized by two bright bands at approximately 4.5 and 1.9 kb, representing 28S and 18S ribosomal RNA, and the absence of genomic DNA.

The integrity of the RNA can be determined by running an aliquot of the RNA preparation on a 1% denaturing agarose gel, or, if the amount of RNA is limited, using the Agilent 2100 Bioanalyzer.

A simple test for genomic DNA contamination is to use your RNA directly as a template in a PCR reaction with primers for any well characterized gene, such as beta-actin or GAPDH. The primer positions should not span a large intron and should be chosen to amplify a short fragment (<1 kb in length). RNA subjected to a reverse transcription reaction can be used as a positive control. If the PCR produces a visible band on an ethidium-bromide stained agarose gel, the RNA preparation contains genomic DNA. For a successful microarray experiment, the genomic DNA content should be less than 0.001%, and should not produce a visible band after 35 PCR cycles.