A comparison of the use of in vitro-transcribed and native rRNA for the quantification of microorganisms in the environment

Nearly full-length, small subunit (SSU) rRNA was transcribed in vitro from clones of SSU rDNA genes. Comparing the use of in vitro-transcribed and nat ive rRNA indicated that, when in vitro-transcribed rRNA was used as a stand ard for quantitative hybridizations with oligonucleotide probes, the popula tion was consistently underestimated. The population abundance was expresse d as a percentage of specific target SSU rRNA (determined with a specific o ligonucleotide probe), relative to the total SSU rRNA (measured with a univ ersal probe). Differences in hybridization signals could be related to spec ific probe target locations and rRNA denaturation conditions, suggesting th at higher order structure is important in quantitative membrane hybridizati ons. Therefore, in vitro-transcribed rRNA cannot always be used for the abs olute quantification of microbial populations, but can be employed as a sta ndard to quantify shifts in population abundance over time, and to compare community structure in various environments.