Perturbation-based gene regulatory network inference to unravel oncogenic mechanisms

The project consisted of single and double knockdown experiments involving forty-five genes in the human squamous carcinoma cell culture line A431. During the knockdown phase, the cells were transfected with siRNAs which decreased target gene expression. After 72 hours the cells were deemed to have reached a new steady-state and approximate cell counts were calculated using resazurin. At this point the cells were lysed using CelluLyser, and a proprietary RNA spike was added to each sample relative to the cell count to act as a reference gene for the qPCR analysis. Then cDNA was prepared from the RNA and preamplified in preparation for the high-throughput qPCR screening. The transcript profiles, with respect to the forty-five target genes, were determined using TaqMan assays on Fluidigm's Biomark platform using 96x96 Dynamic Array IFCs. 384 experiments, 192 technical replicates, Experiments are replicates on the first 3 plates