A study of 16S rRNA for bacterial gene expression analysis

Method: In order to estimate the copy number of each gene, individual calibration curves were generated with the recombinant plasmid for each inserted gene. Gene expression of 16S rRNA, Kgp, RgpA, DnaK, Sod, Dps, PG35, PrtC, Tpx, and OxyR was investigated by qRT-PCR using different growth-stage samples in early-log (EL), mid-log (ML), late-log (LL), and early stationary (ST-E) phases.

Result: The copy numbers of 16S rRNA in 0.1 ng of total RNA in EL, ML, LL, or ST–E was 6,920,000, 8,983,000, 9,798,000, or 11,218,000, respectively. The transcript was observed to slightly increase during growth. The average number of mRNAs for Kgp, RgpA, DnaK, Sod, Dps, PG35, PrtC, Tpx, and OxyR in all the growth-phase samples was 50,273, 44,357, 5,535, 3,876, 2,414, 1,803, 870, 541, and 166, respectively. The copy numbers of their genes ranged from 1/10,000 to 1/100 in comparison with that of 16S rRNA.

Conclusion: The expression of 16S rRNA may be unstable in some experiments. Even if the 16S rRNA expression changes are stable among the samples, the copy number of 16S rRNA may be significantly different from that of the inducible genes. Therefore, it may be necessary to choose stable genes expressing copy numbers that are similar to the inducible target genes between samples or use external controls, especially for normalization when performing a microarray analysis.