EpiQ™ Chromatin Analysis Kit

Chromatin structure data from the EpiQ chromatin analysis kit strongly correlate with gene expression levels for several genes in various cell lines. A, the chromatin state of the proximal promoter regions for four constitutively expressed genes (GAPDH, ACTB, TBP, and B2M) was assessed in four different cell lines (HeLa, PC3, LNCaP, and HCT15). Each gene promoter was determined to be in an open chromatin structure, consistent with medium to high levels of constitutive mRNA expression determined by qPCR. B, eleven epigenetically regulated gene promoters (ABCB1, CDH1, CDH13, DAPK1, DKK1, GSTP1, PTGS2, TP73, WT1, p14ARF, and p16INK4A) were evaluated for chromatin structure in the same four cell lines. Chromatin state varied across the panel of genes and strongly correlated with the gene expression levels. Promoter regions in an open state were associated with medium to high levels of gene expression (shown in the black box, upper right), whereas those in a closed state were linked to low expression (lower left). The EpiQ kit easily identified one outlier gene in PC3 cells (black arrow, •), which exhibits a rare form of epigenetic regulation — monoallelic methylation — where only one allele is methylated, resulting in 50% chromatin accessibility and high levels of gene expression.

The EpiQ chromatin analysis kit utilizes nuclease accessibility to discriminate open vs. closed chromatin regions. Amplification of proximal promoter regions for the epigenetically silenced HBB (reference) gene or the constitutively expressed GAPDH (target) gene was carried out in HeLa cells using the EpiQ kit and EpiQ™ chromatin SYBR^® Green supermix on the CFX96™ real-time PCR detection system. A, closed chromatin regions were protected from nuclease digestion and remained intact prior to amplification, resulting in minimal quantification cycle (Cq) delays (ΔCq = 0.58) following nuclease treatment; B, open chromatin regions were susceptible to nuclease digestion and were unavailable for amplification, leading to significant Cq delays (ΔCq = 8.08) after nuclease treatment. A comparison of ΔCqs with the amplification efficiencies for each gene target factored in was used to determine the accessibility of the target gene, calculated to be >99% for GAPDH. RFU, relative fluorescence units.