Twenty-eight genes were chosen by function of interest and high differential expression by microarray analysis for confirmation of those results by quantitative real-time PCR (qPCR).
Finally, using cDNA microarray analysis and confirmation at protein levels, we observed that the U87 GBM cells that were galectin-1 deficient by means of an antisense galectin-1-stable transfection displayed increased protein levels for p21waf/cip1, cullin-2, p53, ADAM-15 and MAP-2 (12).
The 32 genes examined by qPCR are only a subset of the changes observed in the microarray analysis since qPCR confirmation of microarray changes (2401 genes) was not logistically feasible.
KAK carried out cell line studies, microarray analysis, real-time PCR confirmation and colony formation assays; she participated in study design/coordination and drafted the manuscript.
A set of tissue-specific expressed genes identified from the microarray analysis were selected for confirmation using semi-quantitative RT-PCR.
While not comprehensive of all the genes observed with differential expression in the microarray analysis, the extensive qPCR confirmation experiments validate the quantitative accuracy of the discovery experiments.
A total of 27 targets from the microarray analysis were chosen for confirmation analysis including 15 from the Not Normalized, 5 from the Partially Normalized and 7 from the Normalized groups.
Thirty two genes significantly altered in the microarray analysis were subjected to qPCR confirmation.
In addition to these genes, three non-changing genes (RAP1, MRPS6 and PSMD1) were also selected for the confirmation of microarray analysis.
In the current study, one gene without known annotation but identified as very highly differentially expressed by microarray analysis was chosen for further confirmation by qPCR along with others that did not easily fit into a functional category.
