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Number Determination: Materials and Methods
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
DNA Maxi Kit
the same strain. Before hybridization, genomic DNA was fragmented
into 0.3-2 kb fragments by DpnII
restriction digestion followed by a purification
To determine the mRNA copy number, total RNA (15
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
by random-primed Bioprime
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.
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
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)
Iyer, K. Struhl, Proc Natl Acad Sci U S A 93,
R. Pollack et al., Nat Genet 23, 41-6.
M. Hereford, M. Rosbash, Cell 10, 453-62.
E. Velculescu et al., Cell 88, 243-51.