Losses to the plants’ canopy caused by the native community of herbivores were measured as the total plant area damaged

ted from Mock-IP ChIP reactions. PCIA extracted DNA was used because it allowed for the degree of concentration required to achieve the mass range desired in the reference DNA dilution series. No detectable assay variability was apparent when a linear dilution series of sheared DNA from a single chromatin preparation was assayed multiple times or when dilution series from multiple chromatin preparations were assayed individually to assess the effect of Dehydroxymethylepoxyquinomicin price shearing event. From these results, we conclude that a single preparation of V DNA, generated from either source, can be used as a reference standard in multiple PicoGreen assays against which ChIP DNA samples of unknown concentrations and from multiple experiments may be compared. Quantitative real-time PCR on quantified ChIP DNA Normalizing ChIP samples to DNA concentration ensures that rtPCR is performed on the same mass of precipitated DNA from specific, positive and negative control IP reactions as well as from the Total DNA control sample. At the very least, including such normalization will minimize assay variation associated with differences in background fluorescence that exists when SYBR Green is used to perform rtPCR. In addition, we predict that DNA normalization may also unmask hidden treatment effects or eliminate data artifacts. To determine whether performing rtPCR on a specific mass per reaction or on a specific volume of the ChIP sample per reaction affected the final rtPCR data or its interpretation, chromatin was prepared from two experiments in which rat SMC were cultured with PDBF-BB or its vehicle for 24 h then ChIP was performed with anti-SRF or anti-AcH4 antibodies. Chelexextracted ChIP and Total DNA samples were then quantified using the PicoGreen assay. The CArG-box region of rat ACTA2 was amplified using either 1 ng/reaction or 6.25 ml/reaction. Based on previously published data, we predicted that PDGF-BB would cause a decrease in SRF binding to ACTA2 promoter CArG boxes concomitant with a decrease in histone 4 acetylation because PDGF-BB suppresses ACTA2 gene expression. In the first ChIP experiment, a 40% decrease in SRF interaction with the ACTA2 promoter was detected regardless of whether DNA normalization took place. Likewise, no effect of DNA normalization was apparent when rtPCR was performed on anti-AcH4 ChIP DNA. However, in the second experiment, quantifying the ChIP DNA unmasked a greater magnitude of change in SRF interaction with the ACTA2 promoter and completely reversed the interpretation of how PDGF-BB affects the level of ACTA2-associated Histone 4 acetylation following treatment of SMC with PDGF-BB. DNA normalization also decreased variability in the MockIP data: across these two experiments enrichment values ranged from 0.16 ” 7 October 2011 | Volume 6 | Issue 10 | e26015 Quick ChIP and Sheared DNA Quantification Assays 0.25 with a coefficient of variation of 19.4%. In contrast, when PCR was performed on 6.25 ml of MockIP DNA, the range of relative enrichment was 0.01 0.34 with a coefficient of variation of 112%. Given the dramatic impact that DNA normalization had on the results of the second rtPCR experiment and on the variability of the MockIP negative control, we recommend using quantified DNA prior to performing rtPCR on ChIP samples. Additional 21123673” assay considerations Cell Harvest for the ChIP assay. The established protocol that we used to harvest cells for the initial experiments presented herein was time consuming and labor intensive. It