( ...)Increased Sensitivity in Microarray Analysis

Transcriptor Reverse Transcriptase in the Eberwine Target Preparation Workflow

Reproducibility and sensitivity are key parameters for the quality of a gene expression profiling experiment. The improved reliability of commercially available microarray platforms has shifted the focus to target preparation as a critical step in microarray analysis. Most of the suppliers of array system platforms recommend the linear amplification of cRNA utilizing T7 RNA polymerase for the synthesis of a labeled target, as first described by van Gelder and Eberwine [1]. With this method, maximum sensitivity can be achieved, especially when starting from a few micrograms of total RNA.

Target Preparation
The first step of this target preparation protocol is the efficient synthesis of cDNA with an oligo(dT)-T7 primer containing a specific T7 promoter sequence at the 5 end. This allows subsequent in vitro transcription of the ds cDNA with T7 RNA polymerase, generating singlestranded labeled cRNA as the target for array hybridization. Transcriptor Reverse Transcriptase (included in the Microarray cDNA Synthesis Kit) is optimized for the efficient transcription of long mRNA, resulting in high yields and sensitivity of cRNA targets.

For a direct comparison of sensitivity and reproducibility of target preparation, we have generated target triplicates from two different human leukemia research samples using (a) the Roche Applied Science (RAS) Microarray Kits and (b) reagents and protocols from Supplier A (Figure 1). The biotin-labeled cRNAs were hybridized to HG-U133A GeneChip arrays from Affymetrix according to the manufacturers protocols. Data anal
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