RNA isolation and was performed using the RNeasy mini kit (Qiagen). RNA was subsequently DNase treated with TURBO DNA-freeTM (Ambion) and precipitated overnight. The RNA was dissolved in RNAse free water and quantified using a NanoDrop spectrophotometer (Nanodrop Tech. Inc., Wilmington, DE).
Label
Alexa Fluor 555
Label protocol
cDNA synthesis was performed using the SuperScriptTM Plus Indirect cDNA labeling System (Invitrogen) as per the manufacturer’s instructions using 20 μg of total RNA. Total cDNA was labeled overnight with Alexa Fluor 555 or Alexa Fluor 647 (Invitrogen)
RNA isolation and was performed using the RNeasy mini kit (Qiagen). RNA was subsequently DNase treated with TURBO DNA-freeTM (Ambion) and precipitated overnight. The RNA was dissolved in RNAse free water and quantified using a NanoDrop spectrophotometer (Nanodrop Tech. Inc., Wilmington, DE).
Label
Alexa Fluor 647
Label protocol
cDNA synthesis was performed using the SuperScriptTM Plus Indirect cDNA labeling System (Invitrogen) as per the manufacturer’s instructions using 20 μg of total RNA. Total cDNA was labeled overnight with Alexa Fluor 555 or Alexa Fluor 647 (Invitrogen)
Hybridization protocol
Equal amounts (100-150 pmol) of labeled cDNA were combined plus hybridization buffer (2X = 50% formamide, 10X SSC, 0.1% SDS). cDNA mix was denatured at 95oC and hybridized 16-18 hours at 42oC to DNA microarray slides which had been prehybridized for at least 30 min at 42oC in 1% bovine serum albumin, 5X SSC (1X SSC is 0.15 M NaCl and 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS), washed in water and dried. Following hybridization the slides were washed sequentially in: 2X SSC + 0.1% SDS for 5 min at 42oC, 2X SSC + 0.1% SDS for 5 min at room temperature, 2X SSC for 5 min at room temperature, 0.2X SSC for 5 min at room temperature, and finally dipped in water and spun until dry.
Scan protocol
Arrays were scanned using a GenePixTM 4000B array scanner (Axon Instruments, Inc.) Raw data files were produced from the scanned images using the GenePix Pro 4.0 software package (GPR files).
Data processing
Red/green fluorescence intensity values were normalized such that the ratio of medians of all features was equal to 1
