mRNA-based parallel detection of active methanotroph populations by use of a diagnostic microarray

Abstract

A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.

FIG. 1.

Methanotroph community analyses using environmental DNA and mRNA. Results of individual microarray experiments were first normalized to positive control probe mtrof173 and then to the reference values determined individually for each probe (see reference 15) and displayed using GeneSpring software. In essence, a value of 1.0 indicates maximum achievable signal for an individual probe, while a value of 0.1 indicates that about 10% of the total PCR product hybridized to that probe. Color coding is indicated on the side bar. M.monas, Methylomonas; M.bacter, Methylobacter; M.microbium, Methylomicrobium; M.caldum, Methylocaldum; M.cystis, Methylocystis; M.capsa, Methylocapsa.

FIG. 2.

Quantitative PCR results. (A) Number of target DNA/mRNA molecules per gram of soil. (B) Relative abundance of type Ia and type II target molecules expressed as a percentage of the sum of type Ia and type II molecules.

FIG. 3.

Methanotroph community analyses using environmental DNA and mRNA. Results of individual microarray experiments were first normalized to positive control probe mtrof173 and then to the reference values determined individually for each probe (see reference 15), averaged between parallels (three parallel lysimeters), and displayed using GeneSpring software. In essence, a value of 1.0 indicates maximum achievable signal for an individual probe, while a value of 0.1 indicates that about 10% of the total PCR product hybridized to that probe. Color coding is indicated on the side bar. M.monas, Methylomonas; M.bacter, Methylobacter; M.microbium, Methylomicrobium; M.caldum, Methylocaldum; M.cystis, Methylocystis; M.capsa, Methylocapsa.