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Perform Microarray Analysis with Limited Bacterial RNA

ription reaction. Total RNA isolation methods that include a glass fiber filter based purification step, such as Ambions RiboPure-Bacteria Kit, give higher aRNA yields than organic extraction methods. RNA purity and intactness can also have a significant impact on amplification. Figure 2 shows typical aRNA yields for 5 bacterial species using 100 ng of total RNA isolated with the RiboPure-Bacteria Kit and amplified using MessageAmp II-Bacteria and a 6 hour IVT reaction. Even with a short 6 hour IVT incubation time, 100 ng of input total RNA results in sufficient aRNA for several replicate array experiments. A 14 hour IVT incubation is recommended when <100 ng of input RNA is used.

Figure 2. MessageAmp II-Bacteria aRNA Yields. MessageAmp II-Bacteria yields of aRNA (g) for 5 different bacterial species. Yields are averages from triplicate, 6 hour in vitro transcription reactions using 100 ng total RNA. E. coli was tested with both 100 ng and 10 ng total RNA input. aRNA concentrations were measured by UV absorbance.


Whole Genome Microarray Analysis
Affymetrix E. coli Antisense Whole Genome Arrays were used to compare biotinylated MessageAmp II-Bacteria aRNA derived from E. coli total RNA and MICROBExpress enriched mRNA, as well as the recommended Affymetrix labeling protocol using 10 g E. coli total RNA. The replicate GeneChip arrays shown in Figure 3 demonstrate high reproducibility for the MessageAmp II-Bacteria technology and the Affymetrix recommended labeling protocol. Pearsons Correlation Coefficients for
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