To evaluate the absolute quantification of a target gene transcription in engineered lactic acid bacteria, we developed the Real-time RT-PCR based on DNA subtraction. We isolated the total RNA from the bacteria samples by glass bead, and then analyzed the Ct data of real-time RT-PCR by DNA subtraction assay. Using this method, we successfully estimated the expression level of CBHII gene in the strain of genetic engineered Lactococcus lactis. Since this method could avoid the mRNA copy number loss, it could be used to estimate the expression of other genes in lactic acid bacteria.
DNA, Bacterial ; genetics ; Gene Expression Regulation, Bacterial ; Genetic Engineering ; methods ; Lactic Acid ; metabolism ; Lactococcus lactis ; genetics ; metabolism ; Organisms, Genetically Modified ; RNA, Bacterial ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods