NifH detection/quantification (qPCR, RT-qPCR; nifH database): Difference between revisions
Created page with " * Page authors: Christian Furbo Reeder, PRIMO * Responsible curator: Christian Furbo Reeder ---- __TOC__ <div class="model-box"> {| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' gene quantification |- | '''Context:''' ''Molecular'' |- | '''Spatial scale:''' |- | '''Temporal scale:''' Days, Seasons |- | '''Units:''' gene copies, transcripts |- | '''Community captured:''' d..." |
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* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]] | * [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]] | ||
Latest revision as of 11:34, 11 February 2026
| What is being measured in 1 - 3 words |
|---|
| Approach: gene quantification |
| Context: Molecular |
| Spatial scale: |
| Temporal scale: Days, Seasons |
| Units: gene copies, transcripts |
| Community captured: diazotrophs (potential or active) |
| Co-measurements: |
Method Overview
Quantification of nifH is typically performed using quantitative PCR (qPCR) to measure nifH gene copy numbers, providing an estimate of diazotroph abundance. Reverse transcription qPCR (RT-qPCR) targets nifH transcripts, enabling assessment of gene expression and thus potential nitrogenase activity
Output
- nifH gene copy numbers (qPCR)
- nifH transcript abundance (RT-qPCR)
- Gene- or transcript-based estimates of nitrogen fixation potential
Scale of measurement
- Molecular / gene-expression level
- Quantitative
- Community-integrated signal
Data generated
- Ct (cycle threshold) values
- Standard curves for absolute quantification
- Gene or transcript copy number estimates
- Metadata-linked abundance or expression tables
Units & currency
- nifH gene copies L−1 or g−1
- nifH transcripts L−1 or g−1
- Relative expression (normalized units)
- Fold change (RT-qPCR)
Sample size
- Varies by study design
- Includes biological replication and controls
Repositories & databases
Limitations
- Gene copy number does not equal nitrogen fixation rate
Example Applications & Protocols
Classic examples
Recent applications
Protocols:
- Standard nifH qPCR and RT-qPCR protocols
- SYBR Green or probe-based detection methods
- Custom laboratory SOPs optimized for primer sets and environmental matrices
Common calculations/conversions
- Gene copies = Ct-based quantification using standard curves
- Normalization to volume, mass, or biomass
- Conversion of transcript abundance to relative expression levels