Hagi BucknWise: Created page with "{{BreadcrumbsPrimaryProduction}} * Page authors: PRIMO * Responsible curator: Hagen Buck-Wiese ---- TOC
{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;" ! Functional gene quantification (RT-qPCR / ddPCR) |- | '''Approach:''' quantitative PCR or digital droplet PCR of target genes or transcripts |- | '''Context:''' ''in situ'', incubation, lab |-..."

2026-05-11T23:18:32Z

Created page with "{{BreadcrumbsPrimaryProduction}} * Page authors: PRIMO * Responsible curator: Hagen Buck-Wiese ---- __TOC__ <div class="model-box"> {| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;" ! Functional gene quantification (RT-qPCR / ddPCR) |- | '''Approach:''' quantitative PCR or digital droplet PCR of target genes or transcripts |- | '''Context:''' ''in situ'', incubation, lab |-..."

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{{BreadcrumbsPrimaryProduction}}

* [[Page authors|Page authors]]: [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User:Hagi BucknWise|Hagen Buck-Wiese]]
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{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! 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<sup>-1</sup>
|-
| '''Community captured:''' taxon, group, or community
|-
| '''Co-measurements:''' cell abundance and biovolume (carbon biomass), chemotaxonomic pigments
|}
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== 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<sup>-1</sup>), 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<sup>-1</sup>. The currency is gene or transcript copies.

=== Sample size ===

Typical samples are ~1 L in volume.

=== Repositories & databases ===

== 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 ==

=== Classic examples ===
*

=== Recent applications ===
*

=== Common calculations/conversions ===
* Absolute copies L<sup>-1</sup> = (copies per reaction × reaction volume / template volume) × (extraction volume / sample volume filtered).

== References ==

[[Category:Main Pages|Model types]]

RT-qPCR/ddPCR - Revision history