RT-qPCR/ddPCR

• Page authors: PRIMO
• Responsible curator: Hagen Buck-Wiese

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Functional gene quantification (RT-qPCR / ddPCR)
Approach: quantitative PCR or digital droplet PCR of target genes or transcripts
Context: in situ, incubation, lab
Spatial scale: point sample
Temporal scale: minutes (RNA snapshot)
Units: copies (transcripts) L-1
Community captured: taxon, group, or community
Co-measurements: cell abundance and biovolume (carbon biomass), chemotaxonomic pigments

Method Overview

Water samples are filtered onto polycarbonate or PVDF membranes, and nucleic acids (DNA and/or RNA) are extracted using standardised commercial kits. For transcript-based assays (RT-qPCR), RNA is reverse-transcribed to cDNA. Target functional genes (e.g., rbcL for carbon fixation, 16S/18S rRNA for community composition, psbA for PSII activity) are then amplified using target-specific primers and quantified. In RT-qPCR, the cycle threshold (Ct) is compared to a standard curve of known copy number. In digital droplet PCR (ddPCR), the sample is partitioned into thousands of nanodroplets and each droplet is scored as positive or negative; absolute copy numbers are calculated from the Poisson distribution, without requiring a standard curve.

Both methods can target phylogenetically or functionally specific primers, allowing group-level resolution when combined with group-specific reference genes.

Scale of measurement

Each filtered sample provides a point measurement. RNA extraction captures a snapshot of gene expression at the moment of filtration, reflecting transcriptional activity over the preceding minutes. DNA-based assays represent standing gene pool at time of sampling.

Data generated

Target gene or transcript copy concentrations (copies L^-1), normalised to sample volume filtered. Expression can be normalised to a housekeeping gene for relative quantification of transcriptional changes across treatments or conditions.

Units & currency

Units are copies (transcripts) L^-1. The currency is gene or transcript copies.

Sample size

Typical samples are ~1 L in volume.

Limitations

A single gene copy per genome is assumed; polyploidy or multiple gene copies per genome bias absolute abundance estimates. Gene expression does not necessarily equal metabolic activity, as post-transcriptional regulation can uncouple transcript abundance from protein level and enzymatic function. Diurnal variation in transcript abundance requires time-of-day-standardised sampling. Degenerate primers required for community-level surveys of functional genes (rbcL, psbA) can introduce PCR amplification biases that distort relative abundances.

Example Applications & Protocols

Common calculations/conversions

• Absolute copies L^-1 = (copies per reaction × reaction volume / template volume) × (extraction volume / sample volume filtered).