RNA samples prepared from developing KAX3 cells at 12 hr using TRIZOL(Invitrogen)
Label
cy5
Label protocol
Total RNA (10 µg) was mixed with 1 µg of labeled [dT(18)] primer in 13 µl of water, incubated at 70°C for 10 minutes and on ice for 2-5 minutes. Reaction buffer (Gibco-BRL), 0.1M DTT, 0.5 mM of each dNTP and 200U Superscript II (Gibco-BRL) were added and the reaction was incubated at 42°C for 2 hours. Experimental RNA samples were labeled with Alexa 647 primers and the reference RNA sample was labeled with Cy3. Both of the primers were HPLC-purified by the manufacturer (Operon Technologies). Reverse transcription reactions were terminated with 0.1M EDTA. RNA was degraded by adding 0.3 N NaOH and incubating at 60°C for 20 minutes. The reaction was neutralized with 0.4 M Tris.HCl pH7.6. Labeled cDNA was purified by ethanol precipitation, resuspended in 10 µl of DDW and mixed with 130 µl of PerfectHybTM Plus Hybridization buffer (SIGMA H-7033).
RNA samples prepared from vegetative wild type cells and from 6 developmental times (0, 3, 6, 12, 17, 24 hours) using TRIZOL(Invitrogen)
Label
cy3
Label protocol
Total RNA (10 µg) was mixed with 1 µg of labeled [dT(18)] primer in 13 µl of water, incubated at 70°C for 10 minutes and on ice for 2-5 minutes. Reaction buffer (Gibco-BRL), 0.1M DTT, 0.5 mM of each dNTP and 200U Superscript II (Gibco-BRL) were added and the reaction was incubated at 42°C for 2 hours. Experimental RNA samples were labeled with Alexa 647 primers and the reference RNA sample was labeled with Cy3. Both of the primers were HPLC-purified by the manufacturer (Operon Technologies). Reverse transcription reactions were terminated with 0.1M EDTA. RNA was degraded by adding 0.3 N NaOH and incubating at 60°C for 20 minutes. The reaction was neutralized with 0.4 M Tris.HCl pH7.6. Labeled cDNA was purified by ethanol precipitation, resuspended in 10 µl of DDW and mixed with 130 µl of PerfectHybTM Plus Hybridization buffer (SIGMA H-7033).
Hybridization protocol
Labeled cDNA in PerfectHybTM Plus Hybridization buffer (SIGMA H-7033) was hybridized to the arrays after heat treatment (95°C, 2 min) using a GeneTAC hybridization station (Genomic Solutions) according to the manufacturer's recommended protocol as indicated below.Labeled cDNA was hybridized to the arrays using a GeneTAC automatic hybridization station (Genomic Solutions) for 2 hours at 65°C. Arrays were sequentially washed with 3 solutions, 2x SSC, 0.5% SDS; 0.5x SSC, 0.5% SDS; 0.1x SSC, for 30 sec at room temperature twice each.
Scan protocol
The arrays were scanned with a ScanArrayLite scanner (Perkin Elmer) according to the manufacturer's recommended protocol. PMT and Laser settings were not recorded.All images were processed with the ImaGene software package (BioDiscovery, Inc.).
Description
Reference pool collected at 0, 3, 6, 12, 17, 24 hours from development and labeled with Cy3. Sample co-hybridized with KAX3 strain from 12 hrs of development labeled with Cy5.
Data processing
After quantiation the normalization is implemented in 6 distinct steps: thresholding, quantile adjustment of single channel values, bias adjustment, averaging of on-chip replicates, by-signal-size variance estimation and scaling of the final values using the estimated by-signal-size variance. For more details see Van Driessche, et al.(2005). "Epistasis analysis with global transcriptional phenotypes" Nat Genet 37(5):471-7.
