Affymetrix gene chip hybridization oven 645

Whole genome expression studies were performed by Microarray analysis. Total RNA from untreated and INH treated (20 mM) Hep3B cells was isolated manually by TriZol (Invitrogen). RNA quality and quantity were checked by formaldehyde gel electrophoresis and spectrophotometer respectively. RNA samples with approximately 2:1 ratio of 28 S: 18 S rRNA and 260/280 values ≥1.8 were used for gene expression analysis. First strand was synthesized from total RNA and then second strand was synthesized. Then cRNA was synthesized by in vitro transcription and purified. Further sense strand cDNA was synthesized from cRNA and purified. Now cDNA was fragmented, labeled and loaded on Human Gene 1.0 ST Array Chip and sealed with white tough tag. For hybridization, Chip was transferred to the Hybridization Chamber (Affymetrix Gene Chip Hybridization Oven 645) and incubated for 16 h at 45 °C with 60 rpm speed. After hybridization, Human Gene 1.0 ST Array Chip was washed in wash chamber (Affymetrix Gene Chip Fluidisc Station 450) to remove the unbounded fragmented cDNA. After washing, array was scanned (Affymetrix Gene Chip Scanner) and Microarray Data was analyzed by Gene Spring software and pathway analysis was performed by Ingunity Pathway Analysis (IPA) software from Qiagen. Eight differentially regulated genes were validated by quantitative real time PCR.

The extracted messenger RNA (mRNA) (250 ng) was converted to complementary DNA (cDNA) using GeneChip 3′ IVT Express Kit (Affymetrix, USA) as per manufacturer’s protocol (Affymetrix, USA). Next, antisense RNA (aRNA) amplification was performed using the GeneChip 3′ IVT Express Kit (Affymetrix, USA). After aRNA synthesis, the aRNA was placed on ice and quantified immediately for fragmentation. Hybridization was performed using the GeneChip PrimeView array (Affymetrix, USA) in the Affymetrix GeneChip Hybridization Oven 645 (Affymetrix, USA). After washing and staining, the probe arrays were scanned using the Affymetrix GeneChip Scanner 3000. A total of 3 biologically independent assays were performed.^{12 (link)}