Figure 5

Target validation by Q-PCR of Chromatin Immunoprecipitation (CHIP)-Based Assays. For target validation, female C57BL/6J mice (n = 20 per group) were instilled with 200 μl of one of the following substances: control inactive peptide (LRGILS [94]) or PAR-activating peptides (PAR1-AP = SFFLRN [94]; PAR2-AP = SLIGRL [94]). Twenty-four hours after instillation, bladders were removed and frozen. 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 that was isolated and then purified using phenol extraction and EtOH precipitation. The final CHIP DNAs were then used as templates for Q-PCR reactions using primer pairs specific for each gene of interest, additional material 2 (Table 2). 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 statistical significant difference (p < 0.05).