Figure 9.

Target validation of selected genes and small GTPases by ChIP/Q-PCR. Female C57BL/6J mice (n = 20 per group) were instilled with 200 μl of one of the following substances: BCG (total dose of 1.35 mg) or pyrogen-free saline on days 1, 7, 14, and 21, as described above. Mice were euthanized 24 hours after a single instillation (BCG acute) or 7 days after 4 weekly instillations (Control and BCG chronic). Bladders were exposed briefly to formaldehyde for cross-linking of the proteins and DNA together, followed by sonication to fragment the DNA. An antibody against RNA polymerase II (Abcam) was then used to precipitate the DNA transcriptome. The final ChIP DNAs were then used as templates for Q-PCR reactions using primer pairs specific for each gene of interest (Table S4; (Additional File 4). Q-PCRs were run in triplicate and the averaged Ct values were transferred into copy numbers of DNA using a standard curve of genomic DNA with known copy numbers. The resulting transcription values for each gene were also normalized for primer pair amplification efficiency using the Q-PCR values obtained with input DNA (un-precipitated genomic DNA). Results are presented as "transcription events detected per 1000 cells" for each gene tested. Error bars correspond to standard deviations from the triplicate Q-PCR reactions. Control represents an un-transcribed region of the genome. Asterisks indicate a statistically significant increase (p < 0.05) between BCG-treated and control and a pound sign indicates a statistically significant decrease (p < 0.05) between BCG-treated and control.