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Synthesize High Yields of Biotinylated aRNA

ageAmp II-Biotin Enhanced Kit (MA II-Biotin) or another manufacturers aRNA labeling kit (Kit A). Two IVT incubation times (4 and 14 hrs) were tested with 1000 ng of total RNA input. Amplification of as little as 50 ng total RNA with the MessageAmp II-Biotin Enhanced Kit produced a comparable number of Present calls as 1000 ng input RNA that was amplified with Kit A. Furthermore, the amplification reaction can be completed in one day.


Hybridization of the biotinylated aRNA produced from the MessageAmp II-Biotin Enhanced Kit to Affymetrix GeneChip Arrays was used to assess the sensitivity and reproducibility of the method and reagents. Increased sensitivity can be measured several ways. One indication of sensitivity is the number of Present calls calculated by the GeneChip Operating Software (Figure 3). Additional analysis of the distribution of signal intensities indicates that the new MessageAmp II-Biotin Enhanced Kit produces a higher mean value. This means that a there was a higher signal intensity across the array (Figure 4). This results in more reproducible array results and increased sensitivity of lower expressed transcripts.

Figure 4. Higher Signal Intensity Distribution on Affymetrix Human Genome Focus Arrays. Distributions of log2 signal intensity values were plotted for the MessageAmp II-Biotin Enhanced Kit and another manufacturers amplification kit (Kit A). The median values for each of the MessageAmp II-Biotin Enhanced plots were similar for total RNA inputs ranging from 50 to 1000 ng. In contrast, signal intensities produced by Kit A at the 1000 ng total RNA input level was markedly reduced. The box and whiskers plot ab
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