5uL of 10% BSA and 28.5uL of 2X enhanced hybridization buffer (Genisphere, Hatfield, PA) were added to the sample to make the hybridization mix. This was applied to Invitrogen NCode miRNA microarrays containing 1053 miRNAs from 6 species (467 human miRNAs) printed in triplicate. The arrays incubated overnight (18 hours) in a humidified chamber at 52oC and the following morning, serial 15 minute washes were done with 2x SSC/0.2% SDS at 52oC, 2x SSC at room temperature and 0.2X SSC at room temperature.
Scan protocol
GenePix 4200AL
Description
dye swap
Data processing
Dye swap was included for each sample and both raw data files are available as supplementary files. For each raw data file, the sample labeled with Cy3 is listed first (eg. For '0d_10d_iso080407.gpr' the day 0 sample is labeled with Cy3 and day 10 with cy5). Background subtracted intensities normalized using the Cross-correlation method (Chua et al., 2006) using R scripts. Chua SW, Vijayakumar P, Nissom PM, Yam CY, Wong VV, Yang H: A novel normalization method for effective removal of systematic variation in microarray data. Nucleic acids research 2006, 34(5):e38.
