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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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Real-Time Quantitative PCR: Primer Design, Reference Gene Selection, Calculations and Statistics.

Zhiwei Chen1, Nigel G Halford2, Chenghong Liu1

  • 1Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China.

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Summary

This study outlines a workflow for relative quantitative PCR (qPCR) to accurately measure gene expression. It details key steps like primer design, reference gene selection, and statistical analysis for reliable results.

Keywords:
PCR efficiencynormalized relative quantityquantitative real-time PCRrelative gene expression

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Area of Science:

  • Molecular Biology
  • Genomics
  • Transcriptomics

Background:

  • Real-time quantitative PCR (qPCR) is crucial for nucleic acid quantification.
  • Relative quantification in qPCR is vital for gene expression analysis in functional genomics and transcriptomics.
  • Accurate qPCR requires careful optimization of specific parameters.

Purpose of the Study:

  • To propose a standardized workflow for relative quantitative PCR.
  • To elucidate critical considerations for beginners using qPCR for gene expression studies.
  • To enhance the accuracy and reliability of gene expression measurements.

Main Methods:

  • Designing specific primers for the gene of interest (GOI).
  • Selecting appropriate reference genes or a combination of reference genes.
  • Implementing robust statistical methods for gene expression level calculations.

Main Results:

  • A comprehensive workflow for relative quantitative PCR is presented.
  • Key technical points for accurate gene expression analysis are highlighted.
  • The workflow aims to improve understanding and application for novice researchers.

Conclusions:

  • Mastering specific primer design, reference gene selection, and statistical analysis is essential for accurate relative qPCR.
  • The proposed workflow provides a clear guide for beginners in gene expression studies using qPCR.
  • This work facilitates more reliable and reproducible gene expression data acquisition.