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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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Guidelines for RNA Analysis by Reverse Transcription Quantitative Polymerase Chain Reaction.

Antoine Praité1, Jean-Marie Lambert1,2, Laurent Delpy1

  • 1UMR CNRS 7276, Inserm 1262, Université de Limoges, Limoges, France; CRIBL lab (Control of the B-cell Immune Response and Lymphoproliferations), Limoges, France; Team 3, BioPIC (Biology of Plasma Cells, Immunopathology and Cancer), Limoges, France.

Methods in Molecular Biology (Clifton, N.J.)
|July 23, 2025
PubMed
Summary

This study outlines a detailed method for analyzing gene expression using reverse transcription quantitative polymerase chain reaction (RT-qPCR). It covers RNA extraction, complementary DNA synthesis, and quantitative PCR (qPCR) assays for accurate gene expression analysis.

Keywords:
Cell cultureGene expressionOligo(dT)Quantitative polymerase chain reactionRNA purificationRandom primersReverse transcription

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Area of Science:

  • Molecular Biology
  • Genetics

Background:

  • Gene expression analysis is crucial for understanding cellular functions and disease.
  • Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a key technique for precise gene expression measurement.

Purpose of the Study:

  • To provide a comprehensive methodology for high-quality RNA extraction and subsequent gene expression analysis using RT-qPCR.
  • To detail protocols for complementary DNA (cDNA) synthesis and compare TaqMan and SYBR Green qPCR assays.

Main Methods:

  • High-quality RNA extraction and column-based purification from cell cultures.
  • Complementary DNA (cDNA) synthesis using oligo(dT) and random hexamers.
  • Quantitative PCR (qPCR) using TaqMan and SYBR Green assays for gene expression analysis.

Main Results:

  • A robust protocol for obtaining high-quality RNA suitable for downstream gene expression analysis.
  • Demonstration of effective cDNA synthesis using different primer types.
  • Comparative insights into the performance of TaqMan and SYBR Green qPCR assays.

Conclusions:

  • The described RT-qPCR methodology enables accurate and reliable gene expression analysis.
  • This comprehensive approach serves as a valuable resource for researchers in various biological contexts.
  • The study highlights the versatility and importance of RT-qPCR in molecular biology research.