RNA in samples collected after 72 h of fermentation was isolated
according to procedures (Santos et al., 2008). 2100 Bioanalyser (Agilent
Technologies) was used to analyze the integrity and concentration of
RNA. All RNA samples were diluted to the same concentration. The potential
DNA contamination was removed by digestion with DNase I
(DNA-free; Ambion). cDNA was synthesized by using oligo(dT)18 and
2 μg of total RNA treated with RNase-free DNase I and M-MLV Reverse
Transcriptase. To quantify the differential expression of the B12 biosynthesis
gene in different fermentations, we performed Q-RT-PCR. Amplification
was carried out in 96-well plates in an ABI Prism 7700 (Applied
Biosystems). Fluorescent agent SYBR Green was used for detection.
Reactions were set up by using the SYBR Green Master Mix from the
same manufacturer, following its recommendations. Primers of cobT,
cbiA, and rpsO (Housekeep gene) were listed here according to Santos
et al. (2008).
The specificity of annealing was checked by the melting curve
analysis. Comparisons were made between different culture media.
To determine relative fold differences for each sample, the threshold
cycle (Ct) value was normalized to the Ct value for rpsO and calculated
relative to a calibrator by using the formula 2−ΔΔCT.