Technology, Protocols, and Analysis

Technology

Affymetrix GeneChip high-density oligonucleotide arrays (HDAs) were used in this study. The yeast genome HDAs are described in detail in Wodicka et al.(1997). The arrays can detect as few as 0.1 mRNA molecules/cell; the dynamic range over which detection is accurate is approximately 0.1 - 100 mRNA molecules/cell.

With the Genechip arrays, the entire yeast genome is covered by four HDAs. In total, 6181 ORFs are present within this set. Each gene is represented on the HDA by 20 25-mer oligos that match the sequence of the message (perfect match oligos) and 20 oligos that are identical but differ by one base (mismatch oligos). Expression levels are calculated by subtracting the signal of a mismatch from its perfect match partner and averaging the difference for each oligo pair for a given gene. The average difference value is a measure of the expression level of that gene. Based on various criteria (e.g. consistent behavior of each oligo pair) a Present or Absent call is also returned (more details at Affymetrix and Wodicka et al., 1997).

Experimental Protocol

Two independent experiments were performed for each wild type versus mutant comparison. Individual mRNA levels were scored if the computer algorithm analyzing the scanned results (Wodicka et al., 1997) returned a "Present" call in both the two wild type and the two mutant expression profiles for that gene or if the expression levels of that gene changed in the same direction and were greater than background levels in both wild type and mutant comparisons. A decrease was called if an mRNA dropped more than two-fold in both comparisons.

Expression profiles were determined by growing yeast cultures to mid-log phase, isolating total RNA, spiking in control RNA for normalization and isolating poly-A RNA. This was used to generate double stranded complementary DNA (dscDNA) that in turn was used to generate biotin labeled copy RNA (cRNA; the oligo used for dscDNA synthesis contains a T7 RNA polymerase promoter). 1-3 mg of polyA RNA thus resulted in synthesis of approximately 60 mg cRNA. This was fragmented and hybridized to the oligonucleotide arrays, arrays were washed, stained, washed and scanned.

The protocols included here are set up to monitor transcription factor mutants that result in significant loss of mRNA (e.g. rpb1-1 that results in a 5-fold reduction of mRNA 45 minutes after temperature shift). For mutants that result in less severe loss of mRNA the culture size and RNA preparations can be significantly scaled down and normalization can be performed through assuming that the bulk RNA undergoes no change. The protocols describe how cells were grown and harvested, how total RNA and polyA RNA was prepared. All subsequent steps were carried out exactly as described in the Affymetrix product information supplied with the Genechips (the leaflets are part numbers 700187 Rev 1 and 700163 Rev 1 5/98).

Analysis

Controls
Reproducibility
Genes Scored
Fold Change
Genome Dependence
Assessment of Primary/Secondary Effects