Total RNA was extracted using 3 ml of TRI reagent (Ambion, Austin, TX) per 250-300 mg of tissue from each sample. The extract was treated with 0.02 Units of DNase1 (Ambion) and cleaned up using phenol:chloroform:isoamyl alcohol (25:24:1), a Phase Lock Gel Heavy tube (Eppendorf, Hamburg, Germany) and a YM30 microcon tube (Millipore, Billerica, MA).
Label
cy3
Label protocol
Five micrograms of total RNA was used to synthesize cDNA for each dye using 8 thermal cycles at 52 °C for 10 sec and 44 °C for 15 min. The reaction was stopped with 3.5 µl of 0.5 M NaOH/50 mM EDTA and then heated at 65 °C for 15 min. The solution was neutralized with 5 µl of 1 M Tris-HCl (pH 7.5). Finally, 10 µl of cDNA was purified using a MinElute PCR purification kit (Qiagen, Valencia, CA). The fluorescence dye hybridization steps were accomplished by a modification to the 3DNA array 350 kit protocol (Genisphere Inc., Hatfield, PA).
Hybridization protocol
The microarrays were prehybridized for 1 hr with 0.2% I-Block (Tropix, Bedford, MA) in 1X PBS solution at 42 °C, then were washed with distilled water at room temperature for 10 min and finally were again washed with isopropanol at room temperature for 5 min using a rotary shaker. The arrays were dried by centrifugation for 5 min at 1000g. The cDNA and fluorescence dye hybridization steps were accomplished by a modification to the 3DNA array 350 kit protocol (Genisphere Inc., Hatfield, PA). A total of 20 µl of Cy3 (10 µl) and Cy5 (10 µl) labeled cDNA samples was hybridized to each array at 55 °C in a water bath for 16 hr in a dark humidified chamber. The arrays were then washed for 15 min with 2X SSC/0.2% SDS at 55 °C, for 15 min in 2X SSC at room temperature, and finally washed again for 15 min in 0.2X SSC at room temperature. The arrays were again dried by centrifugation for 5 min at 1000g. Both Cy3 and Cy5 capture reagents were combined with the hybridization buffer and were hybridized to an array for 4 hr at 55 °C in a water bath. The arrays were rewashed and dried as previously described (Galbraith et al. 2004).
Scan protocol
At UMC the arrays were immediately scanned on an Axon Genepix 4000B laser scanner (Axon Instruments, Foster City, CA), while at UMN, the arrays were scanned on a GSI Lumonics ScanArray 5000 laser scanner (GSI Lumonics, Watertown, MA). The image data were extracted using BlueFuse for microarrays (BlueGnome, Cambridge, UK)
Data processing
Spots with a quality score of 0 or with a confidence score of less than 0.1 were removed from the data. The filtered data for each array were log2 transformed and with-in slide normalized by performing a confidence-weighted LOESS regression for each print-tip to correct for an physical effects introduced by the printer head as well as to compensate for other spatial variation across the slides {{40 Smyth,G.K. 2003}}. Scale differences between slides due to variations in scanner settings and sample preparation were corrected for by standardizing intensity values to have a zero mean and unit variance using JMP Genomics (SAS Institute, Cary, NC).
