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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 pairs of primers used in the practicals have been validated beforehand.

All qPCR quantifications are done in triplicates.

Calculation of Amplification efficiency = primer efficiency

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

DNA dilution

——📝🔴—– Benoit: 5/07/2022: Last year, we used a 1:5 dilution, instead of a 1:20. Should we upadate (a) the sentence below and (b) the calculations in the different tables? ——📝—–

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

In theory, the Cq of the Samples should not differ from the Input more than a factor of 4, but I consider that we are OK if we stay in that range (avoid exceeding a Sample>30)

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)

qPCR reactions to do

——📝🔴—– Benoit: 5/07/2022: Last year, we used a 1:5 dilution, instead of a 1:20. Should we upadate (c) the sentences below ——📝—–

Figure - Layout for the qPCR
qPCR reactions

Analysis of the results:

The analysis and the different calculation can be done using the provided excel file: Excel file

Adjust the Cq of the input:

Adjustment of the Cq_sample :

Where E stands for the primer efficiency.

Adjustment of the Cq_Input (for the same dilution in qPCR):

For each Sample:

In addition, you can also calculate the standard deviation for this enrichment with the following formula:

where

all the formulas will be explained during the workshop