Start with extraction date, storage history, concentration, purity information, and degradation risk. If the RNA has been thawed many times or stored under uncertain conditions, repeat quality checks before using old concentration values.
2. Plan reverse transcription
Record RNA input, primer strategy, reaction volume, enzyme conditions, and whether genomic DNA removal was performed. Include a no-RT control when genomic DNA carryover could change interpretation.
3. Design qPCR controls
Plan NTC, positive control, no-RT control, reference genes, and technical replicates before building the plate. Controls should be visible in the layout, not added as an afterthought.
4. Prepare the master mix
Use one shared master mix when possible and include overage. Keep template addition separate from clean master mix assembly to reduce contamination risk.
5. Interpret carefully
Review curve shape, replicate spread, Ct range, control behavior, and reference gene stability before interpreting ddCt or fold change. A clean calculation cannot rescue weak upstream evidence.
Documentation
- RNA extraction and storage notes.
- Reverse transcription inputs.
- qPCR plate layout and controls.
- Excluded wells and reasons.
- Final interpretation with caveats.