PCR and analysis

Enrico Bagnoli, Miratul Muqit

Published: 2024-05-14 DOI: 10.17504/protocols.io.kxygxypwdl8j/v1

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Abstract

This protocol details the PCR analysis.

Steps

PCR setup

Include at least two housekeeping control

Note

The following steps refers to a 384 well-plates, but volumes can easily be adapted to 96 well plates. Perform calculation while waiting for primers to defrost. Always add a few spare wells to ensure enough master mix is made as Sybr Green is very viscous and difficult to pipette precisely.Use a dedicated set of pipettes and try to be as clean as possible. Ensure primers efficiency is satisfactory. Include wells with no cDNA as amplification control.Run samples in triplicates. Include at least two housekeeping control

Note

If first time, prepare 100micromolar (µM)primer stock by diluting it with PCR-grade water.

Prepare a working solution at 10micromolar (µM) by diluting 1:10 the stock in PCR-grade water.

In each well of a 384 well-plate,5µL of samples + master mix have to be added, according to the following volumes:

DNA: 2µL
MasterMix: 2.5µL SyberGreen + 0.5µL forward and reverse primers working stock

Prepare, for each gene used, a master mix, by adding 2.5µL/well of SYBR green and 0.5µL/well of primer mix in a clean PCR-grade tube.

Dilute stock of cDNA. A 1:5 or 1:10 dilution is normally sufficient to detect most genes and to have enough cDNA for all the samples.

Pipette 2µL cDNA in each well assigned to the same samples.

Pipette 3µL mastermix in each well assigned to the same primer.

When all plate is completed, seal the plate and quickly centrifuge it to prevent bubble interference.

Put the plate in a thermal cycler designed for quantitative measurements.

Note

Conditions of the qPCR: (preamplification step) 95°C for 0h 5m 0s,(amplification step) 45 cycles of 95°C for 0h 0m 10s, 60°C for 0h 0m 10s and 72°C for 0h 0m 20s.Finally, calculate a melting curve to confirm the presence of a single PCR product following these steps: 95°C for 0h 0m 5s, 66°C for 0h 1m 0s, and gradual increase in temperature up to 97°C (fluorescence acquisition).

PCR analysis

The following sections outlined analysis of PCR data using the ΔΔC_t method.

10.

For each sample and for each gene, calculate the average C_t value of the technical triplicate,, checking that intra-replicate variation is acceptable.

11.

For each sample, calculate the geometric mean of the C_t value of the housekeeping genes (C_{t HKG})

12.

For each sample and for each gene of interest, subtract the average C_{t HKG} value (from step 11) from the average C_t value calculated at step 10 to obtain the ΔC_{t GOI} for each sample and for each gene of interest

13.

For each gene of interest, average the ΔC_{t GOI} for the control samples (untreated) across your experimental replicates to obtain the control ΔCt GOI _{t GOI}

Note

Both arithmetic and geometric mean can be used, the latter is particularly appropriate when the ΔC_t values across experimental replicates are quite dispersed.Normalising by averaging across experimental replicates allows to have an error bar also for the control group. If the normalization is performed intra-experiment, all control points will be at 1, without error bars and affecting statistical analysis.

14.

For each gene of interest, calculate the ΔΔC_{t GOI} by subtracting the control ΔCt GOI _{t GOI} from the ΔC_{t GOI} for each sample and for each experimental replicates

15.

At this point, you should have, for each gene of interest and for each sample, n ΔΔC_{t GOI} where n is the number of experimental replicates.

Note

Perform the appropriate statistical analysis at this step, has the pints will most likely be normally distributed. In the last step, the normality will be possibly lost.

16.

In order to plot the result, use the Fold change for each gene of interest by using the following formula for each data point:

FC=2^(-ΔΔC_{t GOI})

PCR and analysis

Abstract

Steps

PCR setup

PCR analysis

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