Copy Number Determination: Materials and Methods

Total RNA was prepared from mid-log phase culture (OD₆₀₀ ~ 0.5) of yeast strain S288c by hot phenol extraction as described previously (1). Genomic DNA was isolated by using the Genomic DNA Maxi Kit (Qiagen) from the same strain. Before hybridization, genomic DNA was fragmented into 0.3-2 kb fragments by DpnII (New England Biolabs) restriction digestion followed by a purification (Qiaquick PCR Kit). To determine the mRNA copy number, total RNA (15 mg) was labeled with Cy5-dUTP by reverse transcription reaction using an anchored oligo-dT primer (5-dT₂₀VN-3), while genomic DNA (200 ng) was labeled with Cy3-dUTP by random-primed Bioprime Labeling Kit (Gibco BRL) with a few modifications (2). The two flouro-labeled probes were mixed and co-hybridized with the cDNA microarray containing ~ 6200 yeast ORFs. The red to green ratio obtained from each gene represent the absolute abundance of each mRNA transcript per gene. Using the assumption that there are about 15,000 mRNA per cell (3, 4), the global transcriptome profiles of yeast were determined.

Eleven independent measurements were carried out to extensively assess the reproducibility and optimal condition of this method. While keeping other conditions constant, one condition was varied at each time in each measurement including: using total RNA isolated from independent colony and culture; using genomic DNA fragmented by different methods (restriction enzymes Dpn II or Rla III, or by sonication); titrating the amount of genomic DNA and total RNA for each hybridyzation by increasing or decreasing the input amount 5-fold, respectively; comparing total RNA samples with or without DNAse treatment; and using klenow or reverse transcriptase for labeling genomic DNA.


(See details in Fig. 1)

References:

1. V. Iyer, K. Struhl, Proc Natl Acad Sci U S A 93, 5208-12. (1996).
2. J. R. Pollack et al., Nat Genet 23, 41-6. (1999).
3. L. M. Hereford, M. Rosbash, Cell 10, 453-62. (1977).
4. V. E. Velculescu et al., Cell 88, 243-51. (1997).