Metabolites (Jun 2023)

Real-Time Quantitative PCR: Primer Design, Reference Gene Selection, Calculations and Statistics

  • Zhiwei Chen,
  • Nigel G. Halford,
  • Chenghong Liu

DOI
https://doi.org/10.3390/metabo13070806
Journal volume & issue
Vol. 13, no. 7
p. 806

Abstract

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Real-time quantitative PCR is a technique that can measure the content of the target nucleic acid sequence of interest in a given sample. It is mainly divided into absolute and relative quantitative methods. The relative quantification is mainly used in gene expressions for functional genomic and transcriptome studies. However, to use this technology accurately, there are some key points to master. First, specific primers need to be designed to ensure amplification of the gene of interest (GOI). Second, the appropriate reference gene or reference gene combination has to be selected. Finally, scientific gene expression level calculations and statistics are required to obtain accurate results. Therefore, this work proposes a workflow for relative quantitative PCR and introduces the relevant points so that beginners can better understand and use this technology.

Keywords