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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 analysis was performed using the Affymetrix platform- specific software (present calls, signal intensity values) and MS Excel for calculation of the correlation factors (r2).

Data Analysis
We first analyzed the present call percentage. The number of present calls equals the number of genes (probe sets) that are detectable on a chip. This number corresponds to the genes being expressed in the particular sample.



A significant increase in the number of present calls was observed for both samples when labeled targets were prepared with the RAS Microarray Kits as shown in Table 1.



Figure 2 shows the distribution of the present calls in more detail. The genes analyzed are those that were detected with all three target replicates, prepared by the indicated workflow. For sample 1, 10,339 genes were found to be present when targets were prepared with the RAS Microarray Kits. Of these, 9,331 were also detected with all three targets prepared by Supplier As workflow.



For sample 2, when targets were prepared with the RAS Microarray Kits, 9,652 genes were detected. 7,887 of these genes were also detected with all three targets when Supplier As workflow had been used.

Using the RAS Microarray Kit workflow for target preparation, we identified 185 genes for sample 1 and 283 genes for sample 2 that could not be detected with any replicate when targets where prepared by Supplier As workflow. Vice versa, only 63 genes for sample 1 and 21 genes for sample 2 were not detected with targets prepared with the RAS Microarray Kits

A higher call rate and a larger number of uniquely detected genes are consistently observed for targets generated with the RAS Microarray Kits, indicating increased sensitivity.

Furthermore, correlation factors were calculated for a pair-wise comparison of the three chips hybridized with target replicates; only genes detected with all three target replicates were included in the calculation (Table 2). For chips hybridized with targets that were generated using the RAS Micorarray Kit workflow, correlation is very good (> 0.99) for all pair-wise comparisons. When targets were prepared with Supplier As workflow the correlation is somewhat lower. The good correlation observed for chips hybridized with targets synthesized with the RAS Microarray Kits demonstrates the high reproducibility of the RAS target preparation procedure.

In summary, our data show that the RAS Microarray Kit workflow for target preparation consistently leads to very sensitive and highly reproducible microarray results.




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