Controls

Various controls were used to monitor the quality of the experiment and to permit accurate comparisons between experiments. We verify that the yield of total RNA obtained from wild type and mutant cells is very similar. Equivalent amounts of total RNA from each strain are used to make target preparations (stable RNAs such as rRNA and tRNA account for approximately 96% of total RNA). Known amounts of five different in vitro transcribed polyA-tagged B. subtilis RNAs are added to the RNA preparation prior to polyA selection (control features on the HDA are designed to detect the levels of each of these RNA species). The amounts of different B. subtilis RNA molecules added to the RNA preparation represent the maximum range of values expected for the levels of various yeast mRNA species. The values of these controls obtained by scanning and returned by the computer algorithm were used to normalize the values for each yeast mRNA in a given experiment. In this manner, the levels of all mRNA species are normalized to a constant level of total RNA from each strain.

Most experiments involve a comparison between the values obtained for a mutant cell and its isogenic wild type counterpart. Additional controls applied to these experiments come from the analysis of duplicate data sets. For example, a comparison is made between the values obtained for the wild type cells in the two independent experiments. Similarly, a comparison is made between the values obtained for the mutant cells in the two independent experiments. Data is used only when the comparison reveals a high correlation between the two data sets from the duplicate experiment.

For any experiment involving a comparison between a mutant and its isogenic wild type counterpart, the HDAs used were from the same lot number.