Highly Consistent ChIP of RNA Polymerase II by Multiple
The EpiTect Chip One-Day Kit Demonstrates a High Degree of Reproducibility.
Four different researchers worked independently to collect duplicate samples of
two million HCT116 cells. Each performed ChIP with the RNA Polymerase II
antibody (RNA Pol II) or control IgG from a Epitect Chip Antibody Kit. The specific enrichment of ChIP DNA was analyzed by qPCR primers for
the GAPDH proximal promoter. The results are expressed as individual percent of
input results (insert table), their average (chart y-axis), and standard
deviation (error bars). The figure shows that multiple researchers can achieve
ChIP-qPCR of Pol II that agrees with only a 4.8 percent coefficient of
Performance: Signal-to-Noise Ratios
Detecting RNA Polymerase II Enrichment on GAPD Promoter
The Epitect Chip System Detects RNA Polymerase II Enrichment at a
Housekeeping Gene Promoter with Expected Signal-to-Noise Ratios.
Triplicate wells of HCT-116 cells were fixed with formaldehyde. Chromatin
was isolated and sonicated in preparation for immunoprecipitation using the
Human RNA Polymerase II Antibody Kit and the One-Day Kit. The resulting
enriched genomic DNA was then was purified and characterized by real-time
PCR with primers for the proximal promoter region of the GAPD promoter and
for an ORF-free region as a negative control. The results for each
immunoprecipitation are expressed as percent of input, or the fraction of
the total input DNA co-immunoprecipitating with RNA Polymerase II.
The immunoprecipitation (IP) with the RNA Polymerase II antibody (RNA
Pol II) brings down much more GAPD proximal promoter than the IP with
control IgG. Neither source
of antibody precipitates a significant amount of a specific genomic DNA
sequence within an ORF-free intergenic region or "promoter
desert" lacking any known or predicted structural genes.
Performance: ChIP DNA Quality
Epitect Chip One-Day Kit Isolates DNA Free of PCR
ChIP One-Day Kit Isolates DNA Free of Inhibitors of Real-Time PCR.
Chromatin was isolated from four million untreated HeLa cells and sheared
by sonication. DNA was then purified from an input DNA fraction (diamonds),
and immunoprecipitations using either control IgG (triangles), anti-p53 (p53 IP, squares), or RNA Polymerase II (Pol2 IP,
circles) using the ChIP One-Day Kit. Different volumes of each DNA
preparation (1.0, 2.0, and 4.0 µL) were then used in appropriate ChIP-qPCR Primers to obtain threshold cycle values to generate a calibration curve.
The fit of the data to a straight line indicates that no inhibitors are
present in the DNA preparations that interfere with real-time PCR. The
results also demonstrate the reliability of the enrichment by the
Epitect Chip One-Day Kit, because the fold of enrichment is independent of
the volumes of template from the immunopreciptated DNA.
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