TFIIB binds active genes in Rin-m rat insulinoma cells. (A) RNA was isolated and cDNA was synthesized using reverse transcriptase. Transcript levels of active (c-Fos and Ins1) and repressed (Fcgr2b and Mycd) genes were measured by quantitative real-time PCR. Levels of cDNA detected (black bars) were determined using a standard curve generated with purified PCR amplicons. No reverse transcriptase controls (white bars) indicate that signal generated was RNA-dependent. The amount of Fcgr2b and Mycd was over 500-fold lower than c-Fos and therefore represents background amplification in the assay. (B) Real-time PCR quantitation of DNA fragments precipitated in a ChIP assay using 3 μg of TFIIB antibodies (black bars), 3 μg of RNAP II antibodies (gray bars), or 3 μg of Gal4 antibodies as a control IgGs (white bars). The level of immunoprecipitated Ins1, c-Fos, Fcgr2b, and Mycd promoters was measured by real-time PCR. The standard curve was derived from PCR reactions using serially diluted input chromatin DNA as the template. Error bars are SEM.