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 25°C. Cells were shifted to 36°C and collected after further incubation for 30minutes.
Extracted molecule
polyA 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 Cy3 with Oligo dT primer.
A single colony of S. pombe cells on a YES plate was inoculated into YES liquid medium. Cells were collected at 5x(the 6th power of 10) cell/ml at 25°C.
Extracted molecule
polyA 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
Cy3
Label protocol
The polyA-RNA (3-6microg) was labeled by CyScribe Post-Labeling Kit (Amasham Pharmacia) with Cy3 with Oligo dT primer.
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 120 microl (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 vegetatively-growing control cells and cells at 30minutes after shift to the restrictive temperature.
Data processing
RNA from vegetatively-growing control cells were labeled with Cy3, and RNA from cells in each experimental condition were labeled with Cy5. Measured fluorescent intensity, I, was corrected as follows to give a corrected intensity, C:C = I-M, (for IA°U¨M+2s), C = I*2s/(M+2s), (for IA°O¨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 was greater than 2s, the values were considered to be effective data. VALUE (Expression 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.
Submission date
Sep 16, 2005
Last update date
Jan 23, 2023
Contact name
Atsushi Matsuda
Organization name
National Institute of Information and Communications Technology
