PCR, RT-PCR and qPCR Application Guide

How PCR, RT-PCR and qPCR work

PCR uses sequence-specific primers and a thermostable polymerase to amplify a defined DNA region. Reverse transcription converts RNA to cDNA before amplification, while qPCR tracks signal during cycling to support relative or absolute quantification.

1. Extract nucleic acid and assess quality and concentration.
2. Select or design primers, probes and reaction chemistry.
3. Set up reactions with contamination controls and appropriate standards.
4. Amplify and assess specificity, efficiency and quantitative performance as relevant.

Experimental planning and controls

Use no-template controls, reverse-transcription controls where relevant, and suitable positive or reference controls. Primer specificity, amplification efficiency and template quality should be considered before interpreting quantitative differences. Follow the selected reagent protocol for exact chemistry and cycling conditions.