A single colony of S. pombe cells on a YES plate was inoculated into YES liquid medium to grow to 5x(the 6th power of 10)cell/ml at 30°C.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated by acid phenol methods described in http://www.sanger.ac.uk/PostGenomics/S_pombe/, and then polyA-RNA was purified by Oligotex-dT30<super>mRNA purification kit (Takara).
Label
Cy5
Label protocol
The polyA-RNA (3-6microg) was labeled by CyScribe Post-Labeling Kit (Amasham Pharmacia) with Cy5 with Oligo dT primer.
Genome DNA was isolated from the S.pombe cell nuclei according to Matsumoto et al., 1987, Mol. Cell. Biol. vol.7 pp 4424-4430.
Label
Cy3
Label protocol
The isolated genome DNA was divided into 3 aliquots. They were digested with AluI, RsaI, and Sau3AI, respectively, and then were mixed. After short DNA fragments (16bp or less) were excluded by CENTRI-SEP Span column (Applied Biosystems) and were labeled with Cy3-dUTP by Oligotailing kit (Rochediagnostics). After once purification with CENTRI-SEP Span column, 1-1.5µg labeled targets were added to the hybridization solution.
Hybridization protocol
The labeled targets were added to the hybridization solution to contain an equal amount of the Cy3 and Cy5, and then were hybridized onto DNA on the microarray, using Genomic solutions GeneTac Hybridization Station, for four hours at 40°C in Genomic solutions GeneTac Hyb buffer 120microl (including 42% formamide ). The slides after hybridization were washed in the following sequence: (1) 2xSSC 0.1%SDS at 40°C for 5min, (2) 0.2xSSC at 25°C for 1min, and (3) 0.1xSSC at 25°C for 1min.
Scan protocol
Microarrays were scanned using an ArraywoRx arrayscanner, and the acquired data was analyzed by a SoftwoRx software (Applied Precision, Inc., Seattle, WA). Fluorescent intensity in each spot circle (150microm diameter) was measured by Softworx tracker.
Description
Comparing between genome DNA (Cy3) and mRNA (Cy5) of wild type haploid.
Data processing
Measured fluorescent intensity, I, was corrected as follows to give a corrected intensity, C:C = I-M, (for I>M+2s), C = I*2s/(M+2s), (for I<M+2s) where M and s are an average and a standard deviation of I of negative control spots for each wave length respectively. When I = M+2s, that is C=2s, it was set to be a detection limit. When C for either Cy3 or Cy5 or both were greater than 2s, the values were considered to be effective data. Copy ratio r' of the each effective detection spot obtained thus was scaled as follows: r'=r-m, r=logR(base is 2), R=(Ccy5/Ccy3), m is an average of r of all effective detection spots. VALUE(E=the copy number of mRNA) was calculated as follows: E=(R'/S)*100,000, r'=logR'(base is 2), S=SUM(R' of all 4932 ORFs), where the total mRNA number in the cell was estimated at 100,000 copies.
Submission date
Nov 11, 2008
Last update date
Jan 23, 2023
Contact name
Atsushi Matsuda
Organization name
National Institute of Information and Communications Technology
