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 little as 0.1 mRNA molecules/cell; the dynamic range over which detection is accurate is approximately 0.1 - 300 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 oligo's that match the sequence of the message (perfect match oligo's) and 20 oligo's that are identical but differ by one base (mismatch oligo's). 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

Wild type and mutant yeast strains were grown to an O.D. 600 of 0.5 in YPD at 30°C. If a temperature-sensitive mutant was under study, the cultures were diluted with an equal volume of YPD at 44°C and allowed to grow for 45 minutes at 37°C. Total RNA was isolated and the genomic expression profile of each strain was determined using four Affymetrix GeneChip arrays. polyA mRNA was isolated, converted to biotin-cRNA, hybridized, washed, stained and scanned as described previously (Wodicka et al., 1997). Five polyA-tagged control RNAs were added to equal amounts of total RNA from each preparation. The levels of these controls were then used to normalize each wild type and mutant expression profile to total RNA. 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 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.

Analysis

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

Back to Genome-Wide Expression Page