Relative and absolute quantitative real-time PCR-based quantifications of hcnC and phlD gene transcripts in natural...

Transcriptional analysis of microbial gene expression using relative quantitative real-time PCR (qRT-PCR) has been hampered by various technical problems. One such problem is the unavailability of an exogenous standard robust enough for use in a complex matrix like soil. To circumvent this technical issue, we made use of a recently developed artificial RNA (myIC) as an exogenous "spike-in" control. Nonsterile field soil was inoculated with various concentrations of the test bacterium Pseudomonas sp. strain LBUM300, ranging from 4.3- to 8.3-log bacterial cells per gram of soil. Total soil RNA was extracted at days 0, 7, and 14 postinoculation, and using two-step TaqMan assays, phlD (encoding the production of 2,4-diacetylphloroglucinol) and hcnC (encoding the production of hydrogen cyanide) gene expression was monitored. For relative quantification, a defined quantity of in vitro-synthesized myIC RNA was spiked during the RNA extraction procedure. Absolute qRT-PCR was also performed in parallel. Both the absolute and relative quantifications showed similar transcriptional trends. Overall, the transcriptional activity of phlD and hcnC changed over time and with respect to the bacterial concentrations used. Transcripts of the phlD and hcnC genes were detected for all five bacterial concentrations, but the phlD transcript copy numbers detected were lower than those detected for hcnC, regardless of the initial bacterial concentration or sampling date. For quantifying a low number of transcripts, the relative method was more reliable than the absolute method. This study demonstrates for the first time the use of a relative quantification approach to quantifying microbial gene transcripts from field soil using an exogenous spike-in control.