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qPCR on ChIPed chromatin

The quality of the immunoprecipitated material is usually evaluated by qPCR before being converted into sequencing library. This is the aim of this section.

Principle of the analysis:

The Bio-Rad CFX Real-Time PCR detection system will be used with two pairs of primers that have been validated beforehand. All qPCR quantifications are done in triplicates.

Here we will use two pairs of primers:

> Pair A
Primers FC77 (ctcccatcaaccccaagagc) and FC78 (gatggagacgtagaaggcgg)
Bind in the middle of Pa_2_11200 CDS, coding for Actin

> Pair B
Primers FC125 (agctactgttgtcgtgctgg) and FC126 (tcgatgaagaatacggcggc)
Bind in the 5’ region of a repressed gene (named Pa_5_80) surrounded with transposable elements

Calculation of Amplification efficiency = primer efficiency

Below is an example of RAW data (output of CFX analysis software)

Using the RAW data, a standard curve can be plotted and the qPCR amplification efficiency calculated as follows:

Calculation of “Percent of Input”

The “Percent of Input” is calculted using the Mean Cq value, primer efficiency and Input dilution. This is illustrated below for two primer pairs and 3 different IPs including one with a control GFP antibody.

Implementation for the practical

Initial dilution of Input and ChIP-ed DNA

You can test different DNA dilutions to come across the right Cq (20-22) or do like me and test 1:5 which, in my opinion, correspondeds to a good approximation of the reality.

In theory, the measured Cq of the Samples should not differ from the ones of the Input by more than 4. The more the Cq differs, the less reliable are the quantifications.

Figure - Initial dilution of the INPUT and ChIP-ed material
dilution

qPCR reactions to do

Figure - Standards preparation
Standards

qPCR mix

Prepare a master mix for n+3 reactions (if you have 20 qPCR reaction to do, the mix should be prepared for 23 reactions)

Figure - Layout for the qPCR using 12-well strips
qPCR reactions
Figure - Layout for the qPCR using 8-well strips
qPCR reactions

Analysis of the results:

all the formulas will be explained during the workshop