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Real Time RT-PCR

Real Time RT-PCR

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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Related Experiment Video

Updated: Oct 2, 2025

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Published on: June 11, 2016

A qRT-PCR Method Capable of Quantifying Specific Microorganisms Compared to NGS-Based Metagenome Profiling Data.

Jinuk Jeong¹, Seyoung Mun^2,3, Yunseok Oh¹

¹Department of Bioconvergence Engineering, Dankook University, Yongin 16890, Korea.

|February 25, 2022

Summary

Quantitative real-time PCR (qRT-PCR) accurately quantifies specific gut bacteria, matching next-generation sequencing (NGS) metagenome profiling results. This method offers a cost-effective alternative for microbial analysis in various industries.

Keywords:

metagenome microbial diagnosis next-generation sequencing quantitative real-time PCR

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Last Updated: Oct 2, 2025

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Published on: June 11, 2016

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Published on: October 15, 2019

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Published on: April 14, 2016

Area of Science:

Microbiology
Genomics
Bioinformatics

Background:

Next-generation sequencing (NGS) is widely used for metagenome profiling, especially for the human gut microbiota.
There's a growing need for quantitative diagnostic technologies for specific microorganisms.
Quantitative real-time PCR (qRT-PCR) remains a valuable technique due to NGS's cost and time constraints.

Purpose of the Study:

To compare the identification and quantification of five bacterial genera using NGS metagenome profiling and qRT-PCR.
To evaluate the accuracy and consistency of qRT-PCR for specific bacterial quantification.
To assess the genus-specificity of developed qRT-PCR primers.

Main Methods:

Comparative analysis of 100 gut microbiota samples using NGS metagenome profiling.
Parallel quantification of five bacterial genera (Akkermansia, Bacteroides, Bifidobacterium, Phascolarctobacterium, Roseburia) via qRT-PCR.
Sanger sequencing for validation of genus-specific primer efficacy.

Main Results:

qRT-PCR assay demonstrated statistically consistent quantification patterns compared to NGS metagenome profiling data.
Genus-specific primers showed high accuracy in identifying and quantifying target bacteria.
Sanger validation confirmed the high genus-specificity of the primers used.

Conclusions:

qRT-PCR is a reliable method for quantifying specific microorganisms in metagenomic DNA samples.
This approach can overcome the limitations of NGS, such as cost and time.
The validated qRT-PCR method offers efficient benefits for microbial industries and diagnostics.