Genomic Insights into the Different Layers of Gene Regulation in Yeast
Figure 1
Outline of the GRO method for yeast cells. Cells grown to the desired condition are sampled in duplicate. One aliquot (arrow pointing rightwardly) is used for run-on labeling with either a radioactive precursor or 11-biotin-UTP. Total extracted RNA is used for macroarray or tiling microarray hybridization. The second cell’s aliquot (arrow pointing downwardly) is frozen and used for RNA extraction and cDNA labeling, with either 33P-dCTP or biotin allonamide triphosphate, and hybridized in a new tiling microarray (BioGRO) or in the same macroarray (GRO) previously used after stripping. This method can be used for single point determination in different strains or can be easily adapted to time-point series after a stress or drug treatment, as shown. RNA pol II molecules are shown in different colors, indicating molecules before the elongation step (yellow), elongating (green), or backtracked (red). Labeled parts of mRNAs or cDNA are drawn in red. RA = mRNA amounts, TR = transcription.
