Genechip sample cleanup module kit

Total RNA was obtained from human Sertoli cells at passage one (P1), passage five (P5) and passage ten (P10) using Trizol reagent (Invitrogen). The concentrations and purity of total RNA were evaluated by measuring the 260/280 nm ratio, and the integrity of total RNA was assessed by 1.0% denaturing agarose gel electrophoresis. Five micrograms of total RNA were reverse-transcribed into single strand cDNA that was subsequently converted to double strand cDNA. The purified double strand cDNA served as a template to synthesize biotin-labeled cRNA using the GeneChip2 WT Terminal Labeling Kit (Affymetrix), and the biotin-labeled cRNA was purified using the GeneChip Sample Cleanup Module kit (Affymetrix) and fragmented to 35–200 bases. The fragmented cRNA was hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays and stained using the GeneChip2 Hybridization, Wash, and Stain Kit (Affymetrix). The arrays were scanned with a GeneChip2 Scanner 3000 7G (Affymetrix), and raw data were analyzed using the GeneSpringGX software.

An ovation Pico RNA Amplification System (Nugen Technologies) was used for the amplification of RNA destined for microarray analysis. RNA amplification of LCM ARC samples used to validate genes identified by microarray analysis was performed using a MegaScript T7 Amplification Kit (Ambion) in combination with the GeneChip sample CleanUp Module kit (Affymetrix). The use of a different method of RNA amplification enhanced the validation of the microarray data.