Methods and Results
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
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
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.
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.
both channels have linear readout range within 20-1000 pg.
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.