Total RNA was obtained by following the protocol provided for the Total RNA Isolation Mini Kit (Agilent Technologies, Palo Alto, CA, USA). RNA concentration and quality were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Delaware, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies), respectively.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ug RNA using the One-Color Low-input QuickAmp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer, and its quality checked by Agilent 2100 Bioanalyzer.
Hybridization protocol
1.65 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent GE hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Yeast Microarrays for 17 hours at 65°C in a rotating hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with Wash Buffer 1 (Agilent) and 1 minute with 37°C Wash Buffer 2 (Agilent).
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides (Scan Region 61x21.6 mm, Scan resolution 5um, R PMT is set to 100% and G PMT is set to 100%, XRD=0.10).
Description
Control 0 mM Run 1
Data processing
The scanned images were analyzed with Feature Extraction Software 10.1.1.1 (Agilent) using default parameters (protocol GE1-v5_10 Apr08) and Grid: 015072_D_20060913) to obtain background subtracted and spatially detrended Processed Signal intensities.
