1. The quality of the RNA is the single most important determinant of success of a GeneChip analysis assay. Specifically, 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 also be differential loss of individual mRNAs.
2. 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's RNeasy cleanup procedure.
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3. 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 agarose gel profile should exhibit a 28S band that is 2 times more intense than 18S ribosomal RNA (Figure-4). It is important that the total RNA is free from genomic DNA contamination There are precautions to be taken while using the RNeasy kit to avoid genomic DNA contamination. We have written Standard Operating Procedures for the isolation of total RNA using both TRIzol and RNeasy methods that are available to all facility users upon request. If genomic DNA contamination is present, it is essential to remove it by DNase treatment. DNase then can be removed by heat inactivation followed by RNeasy cleanup using Qiagen kit.
4. Genomic DNA contamination can be verified by PCR with intron specific primers of a known gene, e.g. actin.
5. The minimum amount of total RNA required for GeneChip analysis is 10 µg. It is best to plan to have 20 µg if possible and avoid repeated freeze-thaw.
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