Affymetrix Arrays

The major potential sources of variability that one encounters in microarray based expression analysis come from non-identical source treatment for like experimental conditions and subsequent sample handling leading up to and including RNA extraction/isolation procedures. In short, maintaining and preserving the integrity of the messenger RNA in the physiological state of the experimental treatment condition is critical to a successful and meaningful analytical outcome. Prevention of RNA degradation starts by utilizing one of two recommended methods for tissues: quick freezing in liquid nitrogen or emersion in RNA Later (Qiagen). Quick freezing in liquid nitrogen is particularly appropriate for larger sized tissue samples (>50mg), as these bigger tissues can be easily visualized/handled and readily ground-up by employing a liquid nitrogen cooled mortar and pestle with negligible material loss during such processing. Smaller tissue samples, such as from needle biopsies or cell culture sources are more suitable to preservation in a solution of RNA Later. Since the mode of action of this product is based on the requirement to penetrate cell membranes in order to inactivate cellular and other contaminating RNAses, it is desirable that the tissues utilized be no greater than 5mm in thickness in any dimension to effectively prevent degradation of internal mRNA. Tissue grinders can be directly employed to homogenize small tissue samples preserved in RNA Later.

Alternatively, when working with tissue culture preparations, one can rapidly pellet and then resuspend the cells in a polarity reducing or chaotropic reagent such as Trizol (Invitrogen) or RLT buffer (Qiagen).  Following suspension in the chaotropic agent, the cellular lysate material may be homogenized using a QiaShredder column (Qiagen) and the total RNA extracted using an RNeasy column (Qiagen).

Regardless of the overall method chosen to extract your total cellular RNA, please be sure to use the same exact isolation approach with all of your sample sources. In addition, a DNAse I digestion step (Ambion’s DNA-free Dnase Treatment Kit) may be performed on the extracted RNA if gel analysis reveals the presence of contaminating genomic DNA. Ultimately, the presence of contaminating DNA can result in inaccurate quantification of your total RNA. Lastly, it is HIGHLY recommended, by both Affymetrix in their manual and by our own direct experience, that your isolation method of choice employ a final column purification step with a Qiagen RNeasy column or equivalent. This final purification step removes remaining dsDNAs, small RNA fragments of less than 200 bases in length, residual organics and/or salts that might interfere with downstream processing steps such as cDNA synthesis or in vitro transcription. Much more consistent and much better yields of labeled cRNA are obtained following the in vitro transcription-labeling reaction when this sample purification step is performed beforehand as a final clean-up.