Basic Applied Bioinformatics

CHAPTER 38Concepts of Real‐Time PCR Data Analysis

RK Choudhary

School of Animal Biotechnology, GADVASU, Ludhiana

38.1 INTRODUCTION

Real‐time quantitative polymerase chain reaction (RT‐qPCR) is used for various applications in basic research, including analyses of gene expression, detection of single nucleotide polymorphism (SNP), and cancer screening. In contrast to conventional PCR, where amplicon is identified by running gel after completion of PCR, in RT‐qPCR, the end production is visualized in “real time” as the product is amplified in the PCR machine. Real‐time detection of amplified product is made possible by the addition of fluorescent dyes in a primer/probe/reaction mixture that reports amplified DNA following each cycle. The intensity of the fluorescent signal is proportional to the amount of template cDNA.

38.2 GETTING STARTED WITH RT‐qPCR

38.2.1 How it works

To understand how RT‐qPCR works, let us consider an example of a sample’s amplification plot (Figure 38.1). Features of amplification plots are:

1. The X‐axis represents the number of PCR cycles, and the Y‐axis represents fluorescence generated from the PCR reaction.
2. Phases of amplification plot comprising two phases:
   • exponential: the amount of qPCR‐product is doubled in each cycle;
   • non‐exponential phase: the reaction reaches a plateau, due to exhaustion of one or more PCR‐ingredients in the reaction mixture.
3. The threshold cycle (C_t) or quantitation cycle (C_q) value: initially, although the product is amplified ...