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 30C. After two washes in EMM2-N (EMM2 depleted of nitrogen sources), cells were transferred to EMM2-N with 0.5microg/ml synthetic P-factor and further incubated at 30C. Cells were collected at each time point after the nitrogen starvation with P-factor.
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was isolated by acid phenol methods described Sanger S. pombe post-genomics website, 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.
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 30C.
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was isolated by acid phenol methods described Sanger S. pombe post-genomics website, 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 40C 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 40C for 5min, (2) 0.2xSSC at 25C for 1min, and (3) 0.1xSSC at 25Cfor 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 (Cy3) and cells at at each time point after nitrogen starvation with P-factor (Cy5).
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. 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
Oct 20, 2005
Contact name
Yuji Chikashige
Organization name
National Institute of Information and Communications Technology
