Normalization: Methods and Results

Normalization is critical in order to directly compare each transcript's abundance at different time points measured by different arrays. For normalization, an internal standard was prepared using a pool of in vitro transcribed Bacillus subtilis mRNAs with artificial polyA tags. PCR products of five B. subtilis genes (ATCC#87482 (Trp), #87483 (Dap), #87484 (Lys), #87485 (Thr), and #87486 (Phe)) were printed onto yeast DNA microarrays (25-125 spots/each array in different locations). In vitro transcribed B. subtilis mRNAs (Ribomax large scale RNA production system-T3, Promega) were pooled as a cocktail at various concentrations of each B. subtilis mRNA. To determine the linear readout range of the spiked-in B. subtilis mRNAs in microarray hybridization, 15 mg total RNA sample were spiked with B. subtilis cocktail A (Trp - 20 pg, Dap - 60 pg, Lys - 200 pg, Thr - 500 pg, and Phe - 1000 pg) and fluorescently-labeled in red (Cy5-dUTP), while 15 mg of the same total RNA sample was spiked with B. subtilis cocktail B (Trp - 1000 pg, Dap - 500 pg, Lys - 200 pg, Thr - 60 pg, and Phe - 20 pg) and fluorescently-labeled in green (Cy3-dUTP). The cocktails A and B represent the minimum and maximum range of yeast mRNA levels, 20-1000 pg, but in a reversed order so that they can also cover a wide range of red to green ratios.

The linear ranges of these spikes in both channels were measured by plotting the average intensity of each B. subtilis transcript as a function of its concentration. As shown in Fig. 1, both channels have linear readout range within 20-1000 pg.

Therefore, prior to labeling and hybridization, and to normalize the decay timecourses, a B. subtilis cocktail was spiked into the total RNA samples of each time point in the decay time course, at a final concentration of 400 pg per 15 mg total RNA. The same cocktail was also spiked into the genomic DNA sample with a final concentration of 400 pg per 200 ng genomic DNA. This allows the arrays to be normalized to a constant level of total RNA for each time point.