The quality of the RNA is the single most important determinant of a successful GeneChip analysis assay. Particularly, differential degradation of RNA can lead to erroneous conclusions about both the relative and absolute mRNA levels in the specimens. Although either mRNA or total RNA can be used as starting material, we prefer total RNA for two reasons:
(1) isolating total RNA is easier and more economical than isolating mRNA and
(2) there is loss of starting material during mRNA purification and consequently more mRNA is required to achieve sensitivity similar to that of the total RNA.
In addition, there may be differential loss of individual mRNAs. We recommend TRIzol reagent for isolation of total RNA from tissue specimens as well as cultured and blood cells. Total RNA isolated using TRIzol should be further purified using the Qiagen RNeasy cleanup procedure. The minimum amount of total RNA required for GeneChip analysis is 5 µg (When RNA amount is limiting, as little as 2 µg of total RNA could be used). The A260/A280 ratio should be at least 1.8 for pure RNA. The quality of RNA should also be assessed by agarose gel electrophoresis. The Bioanalyzer gel profile should exhibit a 28S band that is 2 times more intense than 18S ribosomal RNA (Figure 2). It is important that the total RNA is free of genomic DNA contamination. We have written Standard Operating Procedures for the isolation of total RNA using both TRIzol and Qiagen RNeasy methods. If genomic DNA contamination is present, it is essential to remove it by DNase treatment, a modification included in the RNeasy cleanup protocol.