ChIP-IT® qPCR
Analysis Kit(version A2)
Catalog Nos. 53029

Copyright 2012 Active Motif, Inc.

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
any kind, either explicit or implied. Information may be changed or updated in this manual at any
time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and international
treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2012 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.

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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Components and Storage
ChIP-IT qPCR Analysis Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Protocols – Experimental Set Up
Reagent Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Protocols – qPCR
Section A. qPCR Plate Set Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Section B. Using the Analysis Spreadsheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Section C. Data Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section D. Data Interpretation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Appendix
Section E. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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Overview
Chromatin Immunoprecipitation (ChIP) is a powerful tool for studying protein/DNA interactions,
including transcription factors, co-regulatory proteins, modified histones, chromatin-modifying
enzymes and polymerases. Analysis of individual protein DNA interactions can be determined by
combining ChIP with downstream qPCR analysis. However, there are multiple data normalization
approaches that have been used and the widely used delta CT method, or percent input methodof ChIP DNA qPCR analysis can be difficult to grasp. In order to simplify ChIP qPCR data analysis,
Active Motif has developed a normalization strategy that enables accurate qPCR analysis acrossmultiple samples and experiments.
The ChIP-IT® qPCR Analysis Kit contains standard curve DNA, a standard curve primer pair, positive and negative control PCR primer pairs for human and mouse samples and a qPCR analysis
spreadsheet to perform the analysis calculations. The analysis strategy determines primer
efficiency and utilizes this information in combination with input DNA values, the amount ofchromatin used in the ChIP reaction and the ChIP DNA resuspension volumes to normalize theChIP data.
The ChIP-IT qPCR Analysis Kit contains sufficient reagents to perform 10 standard curves and isrecommended for use in combination with Active Motif’s ChIP-IT® High Sensitivity Kit (Catalog No.
53040). This ChIP-IT qPCR Analysis Kit is designed for use with ChIP DNA from human or mouse
samples. To learn about available ChIP-IT® Kits, species-specific qPCR primer sets or ChIP-validated antibodies, please visit our website at www.activemotif.com/chip.

product

formatcatalog no.

ChIP-IT® qPCR Analysis Kit

10 rxns

53029

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1

Kit Performance and Benefits
ChIP-IT qPCR Standard Kit Advantages:• Simplifies qPCR data analysis to enable a faster, more reliable interpretation of data
• All calculations are included in a simple Excel sheet in which data from your qPCR machine
can be entered directly resulting in automatic data normalization and graph generation• Normalized data can be directly compared across multiple sample types and experiments• Includes human and mouse positive control and negative control qPCR primer sets
• Recommendations for acceptable data values allows users to evaluate the quality of the
ChIP reaction before proceeding with downstream applications such as ChIP-Seq

Comparison of ChIP Kits for Abundant Histone H3K4me3
Binding events detected / 1,000 cells

1600
H3K4me3
IgG

1400
1200
1000

High background &non-specific binding

800
600
400
200
0

Negative GAPDH Negative GAPDH Negative GAPDH Negative GAPDHChIP-IT® HS

Competitor M

Competitor I

Competitor D

Figure 1: ChIP-IT qPCR Analysis Kit was used to normalize data across competitor ChIP Kits.
MCF-7 chromatin was prepared according to each manufacturer’s recommendations for their ChIP assay from 1.5 x 106 cells.
The optimal amount of chromatin for each kit was used in the immunoprecipitation reactions; for ChIP-IT® High Sensitivity(Catalog No. 53040), 10 µg chromatin was used. Following each manufacturer’s protocol, the chromatin was incubatedwith Active Motif’s Histone H3K4me3 antibody (Catalog No. 39915) and a negative control IgG. Following enrichment, qPCR
analysis was performed using the ChIP-IT qPCR Analysis Kit in order to normalize the data and allow for direct comparisonof the results. The included Human Negative Control PCR primer set was used as a negative control as it amplifies a genedesert on chromosome 12 that should not show any H3K4me3 enrichment, while GAPDH is an actively transcribed gene thatis associated with the presence of H3K4me3. Data is expressed as binding events detected per 1,000 cells which represents
the average of the raw data triplicates adjusted for the amount of chromatin in the reaction, the resuspension volume andthe primer efficiency. Because the ChIP-IT qPCR Analysis Kit adjusts for the variation in chromatin amounts and resuspension
volumes, this normalization method allows for direct comparison of data from the different competitor ChIP Kits. This calculation provides consistency in data analysis and allows direct comparison across samples and experiments. To convert thisscale to the percent of ChIP input recovered, divide the values by 1,000. From this data it is apparent that the ChIP-IT HighSensitivity Kit provides at least 2-3 fold enrichment compared to Competitor M and Competitor I, while Competitor D has highbackground and non-specific DNA binding that is visible in the Negative control primer set and IgG results.

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2

Kit Components and Storage
Please store each component at the temperature indicated in the table below.
Reagents

Quantity

Storage

DNA Standard AM1

100 µl-20°C

DNA Standard AM2

100 µl-20°C

DNA Standard AM3

100 µl-20°C

Standard Curve Primer Pair (2 µM)

400 µl-20°C

Human Negative Control Primer Set 1 (2.5 µM)

400 µl-20°C

Human Positive Control Primer Set GAPDH-2 (2.5 µM)

400 µl-20°C

Mouse Negative Control Primer Set 1 (2.5 µM)

400 µl-20°C

Mouse Positive Control Primer Set Gapdh-2 (2.5 µM)

400 µl-20°C

2 mM Tris-HCl, pH 8.0

2 x 1 ml

RT

Additional materials required• Purified ChIP DNA and Input DNA for each sample to be tested• qPCR Primer Pairs to study the gene locus of interest in the ChIP DNA. Positive control
primers are recommended. Primers should be designed to work optimally at an annealing temperature of 58°C. See Reagent Information section for complete details on primer
design.
• 2X SYBR Green qPCR Master Mix (e.g. Bio-Rad Catalog No. 170-8882)• 96-well PCR plate or PCR tubes• Thermocycler

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3

Protocols – Experimental Set Up
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Reagent Information
DNA Standards AM1, AM2 and AM3
The DNA Standards provided in the kit can be used to generate a standard curve. Each standard
vial contains a known amount of genomic DNA. Since the DNA mass in each standard is known,
the number of copies of the genome present in each standard can be calculated (assuming thateach cell contains 6.6 pg of DNA). DNA Standards are provided ready to use. Duplicate qPCR
reactions are prepared for each DNA Standard.
Standard Curve Primer Pair
The Standard Curve Primer Pair is designed to be used with the standard curve generated fromthe DNA Standard samples. The primer pair is provided at 2 µM.
ChIP DNAqPCR reactions with ChIP DNA should be performed in triplicate for each primer pair used. For
ChIP DNA that was eluted in a 100-200 µl volume, use 5 µl DNA per qPCR reaction. However, if
the DNA was eluted in a smaller volume (e.g. ChIP-Seq DNA eluted in 36 µl volume) prepare adilution by adding 6 µl ChIP DNA to 94 µl of 2 mM Tris-HCl, pH 8.0. Use 5 µl diluted ChIP DNA perqPCR reaction. It is necessary to run the included Negative Control Primer Pair (human or mouse)in triplicate with the ChIP DNA. This helps with data interpretation as deviation from the expected
output may indicate potential problems in the ChIP reaction.
Input DNA
Input DNA for each sample type should be diluted to a final concentration of 2.5 ng/µl using
2 mM Tris-HCl pH 8.0. In experiments with multiple Input DNAs prepared from multiple samples,a DNA pool should be made and used for testing. Combine the various Input DNAs at their diluted
2.5 ng/µl concentration into a single tube. Input DNA should be tested in triplicate.
2 mM Tris-HCl, pH 8.0
2 mM Tris-HCl is provided ready to use.
Gene-specific qPCR Primer Pair
When designing PCR primer pairs for use in qPCR analysis of the ChIP DNA, it is recommended
to design primers that generate amplicons 75-150 bp long. Primers should also be designed toperform optimally at an annealing temperature of 58°C with a recommended length of 18-22 bp
each. The Primer3 program (Primer3 at http://frodo.wi.mit.edu/) is a good resource for designing
primers to meet these criteria. We suggest preparing a mix of both primers at a concentration of
2.5 µM each for qPCR reactions. Gene-specific primer sets should be tested with 12.5 ng Input
DNA in a qPCR reaction. Usable primer pairs will have CT values between 23-27. If primers haveCT values greater than 27, the primers should be redesigned.

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4

Human Negative Control Primer Set1
The negative control primer set is designed to serve as a universal negative ChIP control when
performing chromatin immunoprecipitation with human samples. If your test ChIP sample ishuman, run triplicate qPCR reactions using the Human Negative Control Primer Set in addition toa positive gene-specific qPCR primer pair. When the Human Negative Control primer set is usedin the analysis spreadsheet the signal should fall within an expected range. For more information
on data interpretation, please refer to Section D of the manual. The primer pair is provided at 2.5
µM.
Note:

The universal negative control primers could give a positive readout for repressive histone marks such as H3K27me3. In this case we recommend using the
supplied positive control primer set for GAPDH-2 as the negative control.

Human Positive Control Primer Set GAPDH-2
The positive control primer set is designed to serve as a positive ChIP control when performing
chromatin immunoprecipitation with human samples. The Human Positive Control Primer Set
GAPDH-2 amplifies a region of the metabolic gene glyceraldehyde 3-phosphate dehydrogenase(GAPDH) associated with intron 1 and has been validated to work with a multitude of ChIP antibodies including: H3K9ac, H3K14ac, H3K4me2, H3K4me3, H4K5ac, H4K8ac, H4K12ac, H4K16ac,
Total RNA pol II, and RNA pol II phospho Ser5. If your test ChIP sample is human, run triplicate
qPCR reactions using the Human Positive Control Primer Set GAPDH-2 in addition to the genespecific qPCR primer pair for the gene of interest. The primer pair is provided at 2.5 µM.
Mouse Negative Control Primer Set 1
The negative control primer set is designed to serve as a universal negative ChIP control when
performing chromatin immunoprecipitation with mouse samples. If your test ChIP sample ismouse, run triplicate qPCR reactions using the Mouse Negative Control Primer Set in addition toa positive gene-specific qPCR primer pair. When the Mouse Negative Control primer set is usedin the analysis spreadsheet the signal should fall within an expected range. For more information
on data interpretation, please refer to Section D of the manual. The primer pair is provided at 2.5
µM.
Note:

The universal negative control primers could give a positive readout for repressive histone marks such as H3K27me3. In this case we recommend using the
supplied positive control primer set for Gapdh-2 as the negative control.

Mouse Positive Control Primer Set Gapdh-2
The positive control primer set is designed to serve as a positive ChIP control when performing chromatin immunoprecipitation with mouse samples. The Mouse Positive Control Primer
Set Gapdh-2 amplifies the promoter region of the metabolic gene glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) and has been validated to work with a multitude of ChIP antibodies
including: H3K9ac, H3K14ac, H3K4me2, H3K4me3, H4K5ac, H4K8ac, H4K12ac, H4K16ac, Total
RNA pol II, and RNA pol II phospho Ser5. If your test ChIP sample is mouse, run triplicate qPCR
reactions using the Mouse Positive Control Primer Set Gapdh-2 in addition to the gene-specific
qPCR primer pair for the gene of interest. The primer pair is provided at 2.5 µM.
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5

Protocols – qPCR
Section A. qPCR Plate Set Up
We recommend using Active Motif’s ChIP-IT® High Sensitivity Kit (Catalog No. 53040) to performthe chromatin immunoprecipitation. If using a different method to perform ChIP, the enriched
ChIP DNA should be purified prior to use in qPCR analysis.
Following ChIP, qPCR reactions should be performed in triplicate for each primer pair used with
the ChIP and Input samples. The DNA Standard curve should be performed in duplicate using theprovided Standard Curve Primer Pair.
An example 96-well plate qPCR set up including the DNA Standards and standard curve primers
along with six different ChIP DNA samples and four different PCR primer pairs is provided below.
The top table shows the layout of the DNA samples and the bottom table shows the layout of the
PCR primer pairs. Please adjust your plate layout based on the number of samples and primer
sets to be analyzed.
1
A

2

–

3

4

5

6

7

8

9

10

11

12

–

–

–

–

–

Std

Std

Std

Std

Std

Std

AM1

AM1

AM2

AM2

AM3

AM3

B

ChIP 1ChIP 1ChIP 1ChIP 1ChIP 1ChIP 1ChIP 1ChIP 1ChIP 1ChIP 1ChIP 1ChIP 1CChIP 2ChIP 2ChIP 2ChIP 2ChIP 2ChIP 2ChIP 2ChIP 2ChIP 2ChIP 2ChIP 2ChIP 2

DChIP 3ChIP 3ChIP 3ChIP 3ChIP 3ChIP 3ChIP 3ChIP 3ChIP 3ChIP 3ChIP 3ChIP 3

E

ChIP 4ChIP 4ChIP 4ChIP 4ChIP 4ChIP 4ChIP 4ChIP 4ChIP 4ChIP 4ChIP 4ChIP 4F

ChIP 5ChIP 5ChIP 5ChIP 5ChIP 5ChIP 5ChIP 5ChIP 5ChIP 5ChIP 5ChIP 5ChIP 5G

ChIP 6ChIP 6ChIP 6ChIP 6ChIP 6ChIP 6ChIP 6ChIP 6ChIP 6ChIP 6ChIP 6ChIP 6

H

Input

Input

Input

Input

Input

Input

Input

Input

Input

Input

Input

Input

Note: Input DNA contains a mixture of Input material associated with all 6 ChIP reactions.
1
A

2

–

3

4

5

6

7

Standard Curve PCR Primer Pair

8

9

10

11

12

–

–

–

–

–

B
C
D
E

Negative Control

Positive Control

Gene-specific PCR

Gene-specific PCR

Primer Set

Primer Set

Primer Set 1

Primer Set 2

F
GH

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6

1.

2.

Prepare DNA for use in qPCR reactions.
a.

DNA Standards: Use 5 µl DNA Standard per qPCR reaction. Also use 5 µl of the provided 2 µM Standard Curve Primer Pair per qPCR reaction.

b.

ChIP DNA: For ChIP DNA that was eluted in a 100-200 µl volume, use 5 µl DNA perqPCR reaction. However, if the DNA was eluted in a smaller volume (e.g. ChIP-Seq
DNA eluted in 36 µl volume) prepare a dilution by adding 6 µl ChIP DNA to 94 µl 2 mM
Tris-HCl, pH 8.0. Use 5 µl diluted ChIP DNA per qPCR reaction.

c.

Input DNA: Input DNA should be adjusted to 2.5 ng/µl concentration. Use 5 µl per
qPCR reaction.

Set up qPCR reactions as follows:
Reagent

20 µl PCR reactions

2X SYBR Green qPCR master mix

10 µl

PCR primer pair (2.5 µM each)

5 µl

DNA sample (ChIP or Input)

5 µl

Total volume

20 µl

3.

Place the PCR plate in a real time PCR instrument. Using the software for your qPCR machine, assign a value of 3788 to DNA Standard AM1, a value of 378.8 to DNA Standard AM2
and a value of 37.88 to DNA Standard AM3. These numbers represent copy numbers forany unique genomic region of DNA contained in the Standard Curve qPCR reactions. These
numbers are used to calculate copy numbers for the genomic regions detected in the test
reactions on the qPCR plate.

4.

If the gene-specific PCR primer pairs were designed with an optimal annealing temperatureof 58°C, follow the amplification conditions listed below. The provided primers have been
optimized for use with Bio-Rad SYBR Green qPCR Master Mix (Catalog No. 170-8882). Other
master mixes could affect PCR performance and melt curves. If necessary, optimize conditions based on the SYBR Green master mix reagent and PCR instrument used.
95°C for 2 minutes(95°C for 15 seconds, 58°C for 20 seconds, 72°C for 20 seconds) for 40 cycles

5.

Include and inspect the melt curve based on the protocols recommended by the qPCR
instrument manufacturer to ensure that primer pairs amplify only a single product.

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7Section B. Using the Analysis Spreadsheet
1.

Download Active Motif’s ChIP-IT® qPCR Analysis sheet from the Active Motif website atwww.activemotif.com/qPCRanalysis. This file is an Excel template that contains formulas toperform all the calculations necessary to normalize sample data according to primer pair efficiency, the amount of chromatin in each reaction and the resuspension volume of the ChIP
DNA. The spreadsheet template is designed to accommodate data from multiple 96-well
PCR plates in a single analysis.

2.

In column A of the template, fill in the primer name on the first line of each box. Then incolumns B-I, copy the numbers calculated by the qPCR instrument software for Standard
Quantity into the Analysis spreadsheet. In column J, enter the Standard Quantity values for
the Input. (See example below)
A

B

C

D

E

F

G

H

I

J

PrimerSampleSampleSampleSampleSampleSam-

SampleSample

Names

1

2

3

4

5

ple 6

7

8

Negative

Triplicate 1

Triplicate 1

Triplicate 2

Triplicate 2

Triplicate 3

Triplicate 3

Triplicate 1

Triplicate 1

14

Triplicate 2

Triplicate 2

15

Triplicate 3

Triplicate 3

Triplicate 1

Triplicate 1

17

Triplicate 2

Triplicate 2

18

Triplicate 3

Triplicate 3

10
11
12
13

16

Positive

GAPDH

Input

3.

Enter the amount of chromatin used in the ChIP reaction into the template worksheet incell D2. This value should be recorded in micrograms (µg).

4.

Enter the resuspension volume of the ChIP DNA in cell D4. This value should be recorded in
microliters (µl).
For qPCR elution: This is the final volume amount the ChIP DNA was resuspended inat the end of the ChIP reaction (e.g. 200 µl)
For ChIP-Seq elution: For ChIP DNA that was diluted in Section A, Step 1, the final
resuspension volume must be adjusted to account for the dilution factor. Using thevolumes listed in Section A, Step 3 enter 600 µl into cell D4.
(100 µl total dilution volume) / (6 µl ChIP DNA for dilution) = 16.67 dilution factor(16.67 dilution factor) x (36 µl elution volume) = 600 µl equivalent resuspension volume
Note:

This step is critical for accurate quantification of the DNA. If ChIP samples were
not diluted in Section A, Step 1 do not make any calculation adjustments.

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8Section C. Data Calculations
The spreadsheet will perform the calculations to normalize the data based on Binding events
detected per 1,000 cells as follows. The rationale behind the calculations is explained below.
Standard Curvea.

The qPCR instrument will generate a standard curve based on the known quantities
of DNA that were assigned to each DNA standard. The standard curve primer set is
assigned a primer efficiency value of 1.

b.

Based on the values of the standard curve, the qPCR instrument will assign a Standard
Quantity value to each of the ChIP and Input samples. These values are used for thenormalization calculations.

Primer Efficiencya.

Primer efficiency ratios for the gene-specific primers and positive and negative control
primers are calculated by dividing the average input value by the expected copy number in the input. The average input value is the average of the 3 qPCR values that are
generated by the primer set when 12.5 ng of Input DNA are amplified. The expected
copy number is calculated assuming 6.6 pg of DNA per cell.
Input DNA: (2.5 ng/µl) * (5 µl per PCR reaction) = 12.5 ng Input DNA amplified
12,500 pg Input DNA
6.6 pg DNA per cell

* 2 DNA copies per cell = 3,788 copies

Data Normalizationa.

The average of the 3 qPCR values generated by the primer set when each ChIP
sample is amplified in triplicate is calculated. This value represents the number of copies in the 5 µl ChIP sample that was amplified.

b.

The above value is then multiplied by the resuspension volume and divided by 5 to
calculate the number of copies in the entire ChIP reaction.

c.

To normalize the values per 1,000 cells, the value is then multiplied by the ratio of
1,000 divided by cell equivalents in the ChIP reaction.

d.

Finally, the value is divided by the primer efficiency ratio.

e.

The complete formula for normalization is:average qPCR value*(

resuspension volume5) *(primer efficiency ratio

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91000
cell equivalents in ChIP)

Section D. Data Interpretation
The following recommendations are provided to evaluate the success of the ChIP reactions andthe quality of the ChIP DNA. These recommendations are based on the use of the ChIP-IT HighSensitivity Kit (Catalog No. 53040) to perform the chromatin immunoprecipitation reactions. If
another method was used to perform the ChIP reactions, the interpretation results listed below
may not apply. Use the values obtained for Binding events detected per 1,000 cells to performthe data interpretation. See Figure 2 for an example of good and poor results.
1.

2.

3.

Evaluate the signal of the included Negative Control PCR primer seta.

Values should be less than 2 Binding events detected per 1,000 cells. Values higher
than 2 could indicate a poor quality ChIP reaction. The ChIP reaction should be
repeated, especially if the ChIP DNA will be used for ChIP-Seq.

b.

High background levels can sometimes be associated with certain chromatin preparations. If high background persists in ChIP, we recommend making new chromatin.

c.

Some antibodies show signal as high as 1,000 Binding events detected per 1,000
cells. Antibodies that give extremely high signal may have negative control values
above 2. In these cases, values above 2 are acceptable.

d.

The negative control primer may actually serve as a positive control for repressive histone marks such as H3K27me3. If testing a repressive histone, we recommend using
the included GAPDH primer set as the negative control.

Signal strength of the positive control and gene-specific primer setsa.

Antibodies for high abundance targets, such as histone antibodies, will routinely give
signal between 100 and 1,000 Binding events detected per 1,000 cells.

b.

Antibodies for low abundance targets, such as transcription factor antibodies, willroutinely give signal between 10 and 100 Binding events detected per 1,000 cells.

c.

ChIP reactions that give signals between 5 and 10 Binding events detected per 1,000
cells can still perform well in downstream applications such as ChIP-Seq, however, foldenrichment over the negative control primer set becomes the critical variable. If foldenrichments are greater than 5, the ChIP reactions may still be of high enough quality
to perform ChIP-Seq.

d.

ChIP reactions that give signals of less than 5 Binding events detected per 1,000 cells
are usually not of high quality. The ChIP should be repeated or a different antibody
should be used.

Enrichment levels over the negative control primersa.

The negative control primers serve as a measure of the background of the ChIP
reactions and are an important indicator of the quality of the ChIP DNA. The negative
control primers are an alternative to using an IgG or non-specific antibody as a control.
The advantage is that the negative control primers measure the background in the
ChIP reaction containing the actual antibody of interest while the IgG control is a separate reaction that may not have much relevance to what is occurring in the reaction of

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10interest. Fold enrichment refers to signal from positive control or gene-specific primers
divided by signal from negative control primers.

4.

b.

A successful ChIP reaction will have a minimum of 5-fold higher signal with positive
control primers as compared to negative control primers.

c.

When comparing data from multi-sample ChIP experiments it is best not to express thedata as fold enrichment since background levels (measured by the negative control
primer sets) can fluctuate across multiple ChIP reactions. When data is expressed as
fold enrichment, a two-fold increase in background could be interpreted as a 2-foldchange in binding when comparing two samples. Instead, use background levels as ameasure of the quality of the experiment. When comparing multiple samples, changes
in actual binding can be evaluated by directly comparing the Binding events detected
per 1,000 cells.

Conversion of Binding events detected per 1,000 cells to % input
To convert the calculated Binding events detected per 1,000 cells to the more recognizable “% input” scale, simply divide the values by 1,000.

Binding events detected / 1,000 cells

Binding events detected / 1,000 cells

a.

18
16
14
12
10
8
6
4
2
0

18
16
14
12
10
8
6
4
2
0

Poor ualit ChIP D A
ess than -fo denrichment

Cell Line A
Cell Line Bo signastrengthHigh backgroundegative Control primer

Positive Control primer

High ualit ChIP D A
Cell Line A
Cell Line Bo background and morethan -fo d enrichment

egative Control primer

Positive Control primer

Figure 2: Comparison of qPCR results showing good versus poor enrichment over negative control primers.
Data shows qPCR results analyzed using the ChIP-IT qPCR Analysis Kit with the Human Negative Control Primer as a reference and a gene-specific positive control primer. In the top image the negative control primer set for Cell Line A gives highbackground levels with Binding events detected per 1,000 cells above a value of 2, while Cell Line B has positive control
signal levels below 5 Binding events detected per 1,000 cells. The bottom image shows low background in the negative
control primer set with Binding events detected per 1,000 cells below a value of 2. The fold enrichment of the positive control
primer set exceeds 5-fold. Only the ChIP DNA from the bottom sample set is recommended for use in ChIP-Seq.

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11

Appendix
Section E. Troubleshooting Guide
Problem/question

Recommendation

Do I need to run a standard
curve for every primer
pair I include on my qPCR
plate?

No, the included standard curve and primer provide a mechanism by which the efficiencies
of all other primers can be determined. The key is knowing how much DNA is in the standard curve and running qPCR with other primers using input DNA of a known concentration.

Do I have to perform
triplicate reactions?

We recommend triplicate qPCR reactions for ChIP DNA and Input DNA in order to provide
more accurate data. However, the ChIP-IT qPCR Analysis Spreadsheet is able to calculateaccordingly if only single or duplicate values are entered.

When using the provided
Negative Control Primer
Set I saw a positive result.

The included Negative Control Primer Sets are intended to be as universal as possible
and were designed to serve as negative controls for all activating histone marks andtranscription factors. However, these primer sets can give positive results for repressive
histone marks such as H3K27me3. If evaluating repressive histone marks, we recommend
using the supplied Positive Control Primer Set in place of the negative control primer set toestablish a negative result.

Why don’t you recommend
using an IgG control
antibody?

In testing this method across thousands of samples we have found that using primers thattarget an unbound region is a more stringent control than IgG. The negative control primers
serve as an internal control for the ChIP reaction as they are tested using the ChIP DNA
generated from the antibody of interest, while IgG is a completely separate reaction using
an antibody that may give different non-specific binding than the experimental reaction ofinterest.

Why am I detecting negative control primer Binding
events detected per 1,000
cells values greater than 2?

In most cases this means that there is high non-specific binding in your reaction.
Sometimes this can be rectified by repeating the ChIP, but usually it is associated with thechromatin preparation (for reasons that are unknown) and we recommend preparing new
chromatin.
Sometimes antibodies give very high signals and therefore can have correspondingly
higher signal from the negative control primers. If you are achieving enrichment levels of
the positive control primer set of 5-fold above the negative control primers then the higher
background levels are not of critical importance. However, if the downstream application
is for ChIP-Seq, then low background levels are critical. For ChIP-Seq you should strive toobtain less than 2 Binding event detected per 1,000 cells for negative control primers.

Can the Binding events detected per 1,000 cells data
be converted to percent
of input?

Yes, simply divide the output values by 1,000 to achieve percent of input.

Can I use this method with
any ChIP reactions?

This method can be used with commercially available ChIP Kits or homebrew methods tooffer the advantage of normalization based on the amount of starting chromatin used andresuspension volume for direct comparison across experiments. However, our recommendations for specific signal levels and data interpretation are unique for use in combination
with Active Motif’s ChIP-IT High Sensitivity Kit (Catalog No. 53040). If using a different
method to perform ChIP, you will most likely not achieve the low background levels of ChIPIT High Sensitivity and therefore our recommendations on data interpretation and cut-off
signals will not apply.

Can I publish my data using
this method?

Yes, data presented and normalized according to Active Motif’s ChIP-IT qPCR Analysis Kit
protocol has been published many times. To access references, please contact Active Motif.

www.activemotif.com

12Technical Services
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