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

mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly qua...

Full description

Saved in:
Bibliographic Details
Published in:Journal of microbiological methods 2009-11, Vol.79 (2), p.205-210
Main Authors: Dong, Dexian, Li, Junyan, Gao, Qian, Huang, Xianqing, Xu, Yuquan, Li, Rongxiu
Format: Article
Language:English
Subjects:
Bacteriological methods and techniques used in bacteriology
Bacteriology
Biological and medical sciences
Bioreactors - microbiology
Biotechnology - methods
Direct quantification
Fermentation
Fluorescent Dyes - pharmacology
Fundamental and applied biological sciences. Psychology
Microbiology
mRNA
Nucleic Acid Hybridization - methods
Organic Chemicals - pharmacology
Pseudomonas
Pseudomonas - growth & development
Pseudomonas - metabolism
Pseudomonas sp
RNA, Bacterial - analysis
RNA, Messenger - analysis
RNA/DNA hybrid
Staining and Labeling - methods
SYBR Green I
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly quantify mRNA from fermentation samples. After total RNA was extracted, the mRNA was hybridized and protected by a longer DNA oligonucleotide. The probe length determined the strength of signal amplification. S1 nuclease and RNase A were used to remove excess probe, single-stranded RNA, and mis-matched RNA/DNA hybrids. Finally, the perfect-matched RNA/DNA hybrid was quantified by SYBR Green I dye. The conditions of liquid hybridization and enzyme digestion were systemically optimized. The kinetic tendency of phzC mRNA levels during phenazine-1-carboxylic acid fermentation was consistent with the results from MB hybridization in our previous report. The detection of mRNA levels of ten genes in Pseudomonas sp. M18G proved that this method is universal and feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.
ISSN:0167-7012
1872-8359
DOI:10.1016/j.mimet.2009.09.002