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 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.
Dong
Dexian
D
MOE Key Laboratory for Microbial Metabolism, and School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, China. dexian@yahoo.cn
Li
Junyan
J
Gao
Qian
Q
Huang
Xianqing
X
Xu
Yuquan
Y
Li
Rongxiu
R
D052160:Benzothiazoles; D019149:Bioreactors / Q000382:microbiology; D001709:Biotechnology / Q000379:methods; D003959:Diamines; D005285:Fermentation; D005456:Fluorescent Dyes / Q000494:pharmacology; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009930:Organic Chemicals / Q000494:pharmacology; D011549:Pseudomonas / Q000254:growth & development* / Q000378:metabolism*; D011804:Quinolines; D012329:RNA, Bacterial / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods*
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012329:RNA, Bacterial; D012333:RNA, Messenger; C098022:SYBR Green I