Using fluorescent microscopy, we observed that the transfection efficiency of the adenoviral vectors into cells was high and reached more than 95% at an MOI of 50. We selected this group to detect the mRNA expression of HIF-1alpha at different stages by real-time quantitative PCR. The primer pairs were: human HIF-1alpha: sense 5′-CAT CAG CTA TTT GCG TGT GAG GA-3′ and antisense 5′-AGC AAT TCA TCT GTG CTT TCA TGT C-3′. Results show that 60 h after transfection, the expression of HIF-1alpha progestogen antagonist mRNA reach the highest level in the Ad5- HIF-1alpha
group and the lowest level in the Ad5-siHIF-1alpha group. Therefore, for the following studies human NCI-H446 cells were transduced with Ad5, Ad5- HIF-1alpha or Ad-siHIF-1alpha for 60 h at an MOI of 50. Microarray analysis of the gene expression profile of human small cell lung cancer NCI-H446 cells in response https://www.selleckchem.com/products/r428.html to hypoxia by HIF-1alpha To evaluate the effect of HIF-1alpha on gene expression profiles, cells from all 5 groups were harvested for isolation of total RNA, which was used
to synthesize cDNA and labeled cRNA for hybridization to microarrays containing 54,614 gene probes. The experimental protocol was independently performed 3 times. We used the comparative analysis algorithm provided by Genespring to compare differences between the hypoxia group and control group, Ad5-HIF-1alpha group and Ad5 group, Ad5-siHIF-1alpha group and Ad5 group. The genes regulated by HIF-1alpha were determined using a 2.0-fold change cutoff value because this cutoff captured many, but not all of the genes that were previously identified as target genes of HIF-1alpha. We identified 65 gene probes with increased expression (more than 2.0-fold) in the hypoxia and Ad5-HIF-1alpha groups but decreased expression (more than 2.0-fold) in the Ad5-siHIF-1alpha group; 28 gene probes were identified with decreased expression
(more than 2.0-fold) in the hypoxia and Ad5-HIF-1alpha groups but increased expression (more than 2.0-fold) in the Ad5-siHIF-1alpha group (Figure 1B). As supplements for find more the above-mentioned analysis, we performed scatter-graphs of gene chip scanning signals (Figure 1A) and the clustering analysis of gene expression (Figure 1C) to describe the differential expression in response to HIF-1alpha. Figure 1 Microarray and data analysis (A) Scatter graph of gene chip scanning signals: Scatter plot of the normalized microarray datasets resulting from analysis of human SCLC NCI-H446 cells. All 54,614 gene probes are represented in this plot. (B) Experimental design and summary of results: Text in red indicates the total number of genes upregulated in 3 experimental conditions (Ad5-HIF-1alpha vs. Ad5; Ad5 vs. Ad5-siHIF-1alpha; hypoxia vs. control-normoxia). Text in blue indicates the total number of genes downregulated in 3 experimental conditions (same as above).