BMC Genomics 2007, 8:139 http://www.biomedcentral.com/1471-2164/8/139 Quantitative reverse-transcriptase and real-time polymerase chain reactions PC12 cells were NGF-differentiated as above and har- vested at indicated intervals. Total RNA was harvested (Qiagen, Valencia, CA) according to manufacturer's pro- tocol. RNA concentration was determined using a Nano- Drop spectrophotometer (BioRad). Equal amounts of RNA sample were used for SuperScript II first-strand cDNA synthesis (Invitrogen). RT-PCR was carried out using 1 µL of cDNA per sample, HotStart master mix (SuperArray, Frederick, MD) and 0.25 µM each primer (IDT) in a GeneAmp 9700 PCR instrument (Applied Bio- systems, Foster City, CA) for the indicated number of cycles. Equivalent volume of each PCR reaction was run on 2% TBE agarose gels containing ethidium bromide and photographed under UV illumination. Each target was amplified using an appropriate, empirically determined cycle number allowing gel-based visualization of samples within the exponential amplification phase of each reac- tion. Band intensity was measured by integration using ImageJ software [92] and normalized relative to naïve expression value. qRT-PCR expression analysis was performed with cDNA prepared as above in reactions using the Syber Green qRT- PCR master mix (ABI) and analyzed by an ABI Model 7900 Prism instrument. All reactions were carried out in triplicate and measurements analyzed using the relative quantitation 2-∆∆Ct method. The housekeeping gene GAPDH was used as an endogenous control to which sample measurements were normalized. The ROX passive reference dye was used to normalize reaction conditions across sample wells. Naïve PC12 RNA levels for each gene were used as the reference calibrator to which the NGF- treated sample RNA levels were compared. Statistical anal- ysis of qRT-PCR data was carried out using Student's t-test for significance at p ≤ 0.05 comparing treatment to naïve gene expression levels. Authors' contributions CB conceived of the study, coordinated its design, gener- ated cell lines, participated in chromatin immunoprecipi- tation assays, sequence alignment/bioinformatics analyses, molecular/expression analyses and performed data/statistical analyses. PL participated in chromatin immunoprecipitation assays, fragment cloning/sequenc- ing and sequence alignment. BAM conceived of the study and participated in its design and coordinated overall conduct of the study. All authors have read and approved the final manuscript. Additional material Additional File 1 Supplementary Data. A Supplementary data file is included that contains 5 figures and 4 tables. The 5 figures contain additional biological data (Figures 1, 2 and 3) and bioinformatic data (Figure 4) as well as a dia- gram of the ChIP cloning procedure (Figure 5). The tables contain ChIP statistics (Table 1), ChIP primers (Table 2), a list of p53-occupied regions that were non-NGF responsive (Table 3) and RT-PCR primers (Table 4). Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-8-139-S1.pdf] Additional File 2 Cluster Coordinates. A detailed data spreadsheet is included that contains cluster coordinates, chromosome number, and DNA sequences for 212 clusters and roughly 700 associated DNA sequence tags derived from p53 chromatin immunoprecipitation (ChIP) analysis of the rat genome. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-8-139-S2.xls] Acknowledgements This research was supported by the Intramural Research program of the NIH, National Institute of Environmental Health Sciences. 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