Trace genomic DNA in the crude total RNA samples was removed by incubation with 4 10 units DNase I per 100 g selleck kinase inhibitor total RNA at 37 C for 30 min. Total RNA was further purified using an RNeasy Mini kit. The concentration of the total RNA was determined using a NanoDrop ND 1000 spectrophotom eter and RNA integrity was verified using a Bioanalyzer 1000. Generation of Biotin labeled cRNA Biotin labeled cRNA was generated with a modified pro cedure of the Superscript Choice System for double strand cDNA synthesis followed by in vitro transcription. Briefly, the 1st strand cDNA was synthesized from 4. 0 g total RNA by 1. 0 unit SuperScript II reverse transcriptase in the presence of 100 pmoles T7 promoter Oligo dT primer. After 2nd strand synthesis, the DNA was purified with a DNA Clean Concentrator 5 kit and eluted with 8 to 16 l of deionized H2O.

The recovered ds cDNA was further concentrated down to 3 l by a speed vacuum device. cRNA was synthesized with a MEGAscript in vitro Transcription kit. The in vitro transcription reac tion was carried out in a total volume of 23. 0 l consisting of 3. 0 l of ds cDNA, 2. 3 l 10X Ambion reaction buffer, 2. 3 l 10X Ambion T7 enzyme mix, and 15. 4 l NTP labe ling mix. The in vitro transcription reaction was incubated at 37 C for 16 hours in a thermocycler. The cRNA was purified with an RNeasy mini kit. Generally, 40 to 60 g of cRNA can be obtained from 4. 0 g of input total RNA. The size range of the cRNA, expected to be between 300 to 3000 bp with the maxi mum intensity centered at least 1000 bp, was verified using a Bioanalyzer 1000.

The biotinylated cRNA was fragmented to 50 to 200 bp by heating cRNA in a buffer consisting of 40 mM Tris acetate, pH 8. 0, 100 mM potas sium acetate, and 30 mM magnesium acetate at 95 C for 35 min. Oligonucleotide Microarray, Hybridization, Image Acquisition and Data Analysis The bovine microarray platform used was described previ ously. Briefly, a total of 86,191 unique 60mer oligo nucleotides were designed and synthesized it in situ using photo deprotection chemistry. Each unique oligonu cleotide was repeated 4 times on the array. These oligonucleotides represented 45,383 unique bovine sequences genes, including 40,808 Tentative Consensus sequences from TIGR Bos tau rus gene index and 4,575 singletons.

The microarrays were pre hybridized with 1X MES hybrid ization buffer, 40 g herring sperm DNA and 200 g acetylated BSA at 45 C for 15 min followed by hybridiza tion with 10 g denatured and fragmented cRNA per microarray at 45 C for 16 20 h with constant rotation. After hybridization, the microarrays were immediately Carfilzomib washed extensively under non stringent conditions at room temperature fol lowed by a stringent wash at 45 C. After the final rinse with the non stringent wash buffer, the micro arrays were stained with 1�� Stain buffer at RT for 25 min. The stain buffer was removed and the microarrays were rinsed once more with non stringent wash buffer.