cDNA synthesis reverse transcription
Reverse transcriptase converts RNA into DNA (cDNA). For fair qPCR comparisons every sample must be converted under identical conditions — the same RNA amount and the same master mix.
Key points
- Same RNA amount for every sample
- Include a no-RT control to check for genomic DNA
- Choose the primer type for your purpose
Materials
- Quantified RNA
- RT kit (buffer, dNTPs, RNase inhibitor, enzyme)
- Primers (oligo-dT / random hexamers)
- (Optional) DNase
- RNase-free water, PCR tubes
- Thermal cycler
Steps
- 💡 The reagent calculator's "Dilution" tab gives per-sample volumes quickly.
- 💡 25 °C lets random primers anneal; 85 °C inactivates the enzyme.
Calculations
RT master mix (20 µL reaction, plus 10 µL RNA)
🔬 Variable explorer — what changes if…?
The skeleton stays the same, but concentrations, times and reagents change between experiments. Click an item to see what it affects and compare two conditions (A and B) side by side.
RNA input
What it isTotal RNA per reaction.
LessToo little: late Ct and low-expressed genes may be missed.
MoreToo much: the enzyme saturates and inhibitors rise, so conversion stops being proportional.
Typical choice0.1–1 µg per 20 µL (kit range), identical across samples.
Primer type
What it isDecides where reverse transcription starts.
Typical choiceOligo-dT + random mix for routine qPCR.
RT temperature
What it isTemperature at which the enzyme copies RNA.
Lower · lessLower: stalls at GC-rich or folded regions, giving short cDNA.
Higher · moreHigher: standard enzymes lose activity (thermostable ones tolerate 50–55 °C).
Typical choiceThe kit's recommended temperature (usually 42–50 °C).
No-RT control
What it isSame tube without enzyme. Any qPCR signal means genomic DNA is present.
Typical choiceIf no-RT Ct is ≥5 cycles later than the sample, the effect is small (≤ about 1/32).
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| Late Ct overall | Too little or degraded RNA, inhibitors | Re-check quantity and integrity, check A260/230 |
| Signal in no-RT | Genomic DNA contamination | DNase treatment, intron-spanning primers |
| Reference gene Ct varies a lot between samples | Unequal RNA input | Re-quantify, equal input, use a master mix |
⚠ Safety No special hazards, but change gloves often to avoid RNase contamination.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
Analyse the results with free tools:
qPCR analysis (ΔΔCt) → Dilution calculator →