EpiQ™ Chromatin Analysis Kit

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EpiQ Chromatin Analysis Kit

Overview

Schematic Diagram Illustrating Euchromatin and Heterochromatin

The EpiQ™ chromatin analysis kit is a real-time PCR assay for the quantitative assessment of chromatin structure and nuclease accessibility.

Heterochromatin

Heterochromatin
Transcriptionally silent > inaccessible to transcriptional machinery
Inaccessible to nuclease > No DNA digestion > available for qPCR > minimal Cq shift

Euchromatin

Euchromatin
Transcriptionally competent > accessible to transcriptional machinery
Accessible to nuclease > DNA digestion > unavailable for qPCR > large Cq shift

EpiQ chromatin analysis kit process. Epigenetic processes, such as DNA methylation and histone modification, control gene expression by altering chromatin structure. Genes that are actively transcribed are associated with “open” or “accessible” chromatin regions, while genes that are transcriptionally silent are often in “closed” or “inaccessible” chromatin regions. The EpiQ chromatin analysis kit discriminates between open and closed chromatin regions by employing a nuclease accessibility assay and real-time PCR to quantify the level of accessibility.

Manual and Tools for the EpiQ™ Chromatin Analysis Kit

The following manual, guide, and tool will help you obtain optimal results and are required for accurate assay design and data analysis using the EpiQ chromatin analysis kit:

  • EpiQ™ Chromatin Analysis Kit Instruction Manual
  • EpiQ™ Chromatin Analysis Kit Primer Design and qPCR Optimization Guide
  • EpiQ™ Chromatin Kit Data Analysis Tool

1. Getting Started

The EpiQ™ Chromatin Analysis Kit Instruction Manual (part #10019321) is packaged with the product and provides information about the necessary supplies not included in the kit, instructions for use, assessment of assay conditions, experimental protocols, and directions for analyzing data.

2. Design: Before Starting qPCR Experiments

The EpiQ™ Chromatin Analysis Kit Primer Design and qPCR Optimization Guide (Bulletin 6050) provides instructions on how to:

  • Design primers that amplify the promoter regions of target genes
  • Optimize qPCR conditions using the EpiQ™ chromatin SYBR® Green supermix
  • Use pre-existing primers (for example, ChIP primers) for assessing chromatin accessibility with the EpiQ chromatin analysis kit

3. Analysis: After Completing qPCR Experiments

The EpiQ™ Chromatin Kit Data Analysis Tool enables simple and rapid determination of chromatin accessibility from the EpiQ kit. This tool generates three Microsoft Excel spreadsheets based upon user-defined experimental conditions:

  • Data Input is for transferring real-time PCR data
  • Data Summary displays the chromatin accessibility results and EpiQ data quality information
  • Calculations shows the in-process calculations used to determine chromatin accessibility

The EpiQ chromatin data analysis tool requires Microsoft Excel and .NET Framework version 3.5 or higher (for program installation), and a PC running Microsoft Windows XP, Windows Vista, or Windows 7.

EpiQ™ Chromatin Analysis Kit Workflow
top1
top2
Grow cells in 48-well-platesAdd stop bufferPurify genomic DNA (gDNA)Quantitate gDNAAnalyze gDNA using real-time qPCR

Grow cells in 48-well plates
Aspirate media
Add cell permeabilization/
chromatin digestion buffer

Add stop buffer

Purity genomic
DNA (gDNA)

Quantitate gDNA
Prepare reactions

Analyze gDNA using real-time qPCR


EpiQ chromatin analysis kit workflow. Cells grown in culture plates or flasks are treated for 1 hr at 37°C with chromatin buffer in the presence or absence of nuclease. Chromatin digestion is terminated with stop buffer for 10 min at 37°C, followed by genomic DNA purification and quantification. Chromatin structure is assessed via real-time quantitative PCR by comparing results against an epigenetically silenced (reference) gene.

EpiQ™ Chromatin Data Strongly Correlate with Gene Expression Levels

 

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™ Kit Discriminates between Open vs. Closed Chromatin Regions

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.

The EpiQ chromatin analysis kit is a novel epigenetics research tool for the quantitative assessment of chromatin structure in cultured cells. The kit workflow combines in situ chromatin digestion, genomic DNA purification, and real-time PCR for the rapid evaluation of the chromatin environment for targeted regions of the genome. It can discriminate open, actively transcribed chromatin regions from closed, transcriptionally silent regions. The EpiQ kit helps quantify the impact of epigenetic events, such as DNA methylation and histone modification, on gene expression regulation through chromatin state changes.

Key Features and Benefits

  • Short assay time — assessment of chromatin structure in cultured cells can be accomplished in less than
    6 hours
  • Small sample requirement — as little as 5 x 104 cells are required to perform analysis
  • Valuable results — novel assay generates quantitative chromatin structure information with strong correlation to gene expression levels

Manual and Tools for the EpiQ Chromatin Analysis Kit

The following manual, guide, and tool will help you obtain optimal results and are required for accurate assay design and data analysis using the EpiQ chromatin analysis kit:

  • EpiQ™ Chromatin Analysis Kit Instruction Manual — to get started using the kit (part #10019321, packaged with the product)
  • EpiQ™ Chromatin Analysis Kit Primer Design and qPCR Optimization Guide — to optimize design before starting qPCR experiments (Bulletin 6050, only available online)
  • EpiQ™ Chromatin Kit Data Analysis Tool — for analysis after completing qPCR experiments (only available online)

Get more information and download the files from the EpiQ™ Manual and Tools overview.

Applications and Uses

  • Epigenetics research tool
  • Gene expression regulation
  • Chromatin structure assessment

 


EpiQ™ Chromatin Analysis Kit

172-5400
50 preparations, contains components for chromatin digestion, analysis, and genomic DNA purification, and 2x real-time PCR mix for 500 x 20 µl reactions

List Price:   $1,488.00
Quantity:   Add to Quote
This product is not available for sale online. Please contact customer service for more information.
 
EpiQ™ Chromatin Preparation Kit

172-5402
50 preparations, contains components for chromatin digestion, analysis, and genomic DNA purification prior to real-time PCR analysis

List Price:   $625.00
Quantity:   Add to Quote
This product is not available for sale online. Please contact customer service for more information.
 
EpiQ™ Chromatin Preparation Kit

172-5403
100 preparations, contains components for chromatin digestion, analysis, and genomic DNA purification prior to real-time PCR analysis

List Price:   $1,250.00
Quantity:   Add to Quote
This product is not available for sale online. Please contact customer service for more information.
 
(Discontinued) EpiQ™ Chromatin SYBR® Green Supermix

172-5404
5 ml, 2x real-time PCR mix, contains dNTPs, iTaq™ DNA polymerase, MgCl2, SYBR® Green I, ROX passive reference dye, fluorescein, stabilizers, for 500 x 20 µl reactions

This product is no longer available. Please contact customer service for more information on replacement product.
EpiQ™ Chromatin SYBR® Green Supermix

172-5405
10 ml, 2x real-time PCR mix, contains dNTPs, iTaq™ DNA polymerase, MgCl2, SYBR® Green I, ROX passive reference dye, fluorescein, stabilizers, for 1,000 x 20 µl reactions

List Price:   $913.00
Quantity:   Add to Quote
This product is not available for sale online. Please contact customer service for more information.
 
Number Description Options
6252 Reagent Comparison Guide for Real-Time PCR Brochure, Rev C Click to download
6090 Amplification Reagents and Plastics Brochure, Rev D Click to download [ Add to Cart (Free) ]
6020 EpiQ Chromatin Analysis Kit Product Information Sheet, Rev B Click to download
6050 EpiQ Chromatin Analysis Kit Primer Design and qPCR Optimization Guide, Rev C Click to download
10019321 Instruction Manual, EpiQ Chromatin Analysis Kit, Rev A Click to download