Two-microgram of pooled linear amplificated antisence RNA of embryo obtained from pregnant cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5) & cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 & Cy3 fluorescence-labeled cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 micro-l of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 micro-l 1N HCl.The Cy5 & Cy3 cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 micro-g Salmon sperm DNA, 20 micro-g polyA and 20 micro-g yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 10 micro-l, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 15 micro-l with 2.55 micro-l 20XSSC and 0.45 micro-l 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The mixture of labeled probes, for example, the Cy3 & Cy5, were then applied to the microarray, placed a cover slip carefully and incubated at 65oC for 14 - 16 h in a custom slide chamber. After incubation for 14 - 16 h at 65oC, the slides were washed in 2 x SSC / 0.01% SDS for 2 min, followed by washing in 0.2 x SSC/0.01% SDS and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed. Keywords = bovine embryo Keywords = gene expression
