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Related Experiment Video

Updated: Jun 20, 2026

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

Utilizing RNA/DNA hybridization to directly quantify mRNA levels in microbial fermentation samples.

Dexian Dong¹, Junyan Li, Qian Gao

¹MOE Key Laboratory for Microbial Metabolism, and School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China. dexian@yahoo.cn

Journal of Microbiological Methods

|September 15, 2009

Summary

This study introduces a novel SYBR Green I method for precise mRNA quantification in microbial fermentation. This technique offers a universal and rapid approach for monitoring gene expression kinetics.

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Area of Science:

Molecular Biology
Biotechnology
Microbial Fermentation

Background:

Accurate mRNA quantification is crucial for understanding microbial processes.
Quantitative real-time RT-PCR (RT-qPCR) often lacks the required precision for kinetic studies.
Existing methods may not be suitable for direct application to complex fermentation samples.

Purpose of the Study:

To develop a rapid, precise, and universal method for direct mRNA quantification in microbial fermentation.
To enable real-time monitoring of gene expression kinetics for process control.
To overcome the limitations of traditional quantitative real-time RT-PCR.

Main Methods:

Developed a SYBR Green I-based assay for direct mRNA quantification from fermentation samples.
Utilized a protected hybridization approach with a longer DNA oligonucleotide probe.
Employed S1 nuclease and RNase A for selective removal of non-target nucleic acids.
Optimized liquid hybridization and enzyme digestion conditions for signal amplification.

Main Results:

The method demonstrated accurate quantification of mRNA kinetics, exemplified by phzC gene expression during phenazine-1-carboxylic acid fermentation.
Successfully quantified mRNA levels for ten different genes in Pseudomonas sp. M18G, confirming universality.
Achieved results consistent with previous MB hybridization studies, validating the new approach.

Conclusions:

The developed SYBR Green I method provides a precise and universal tool for direct mRNA quantification in microbial fermentation.
This technique has significant potential for real-time monitoring and control of bioprocesses.
The method is broadly applicable across various organisms for gene expression analysis.