Before you start
- Quantify RNA and calculate the volume for the same input in every sample.
- Remove genomic DNA with DNase (kit gDNA-removal step or DNase I).
Reagents and materials
Totals are for 12 reactions (+10% extra). Per-reaction (20 µL) amounts are in the middle column.
| Reagent | Per reaction (20 µL) | Total |
|---|---|---|
| Total RNA (same amount for all samples, 0.1–2 µg) | 1 µg | 1 µg |
| RT buffer / master mix (per kit) | 4 µL | 4 µL |
| Primer mix (oligo(dT) + random hexamers) | 1 µL | 1 µL |
| Reverse transcriptase | 1 µL | 1 µL |
| Nuclease-free wateradjust to 20 µL total | 10 µL | 10 µL |
Equipment
- Thermocycler
- PCR tubes
- Ice
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Remove gDNA
Run the kit’s gDNA-removal step or DNase I treatment, then inactivate as instructed.
Assemble reactions on ice
Combine RNA, buffer, primers, enzyme and water to 20 µL. Prepare a no-RT control (no enzyme) for each RNA or a pool.
Prime
Incubate 25 °C for 5–10 min (random-primer annealing).
Reverse transcribe
Incubate 42–50 °C for 15–60 min, depending on the enzyme.
Inactivate
Heat at 85 °C for 5 min, then hold at 4 °C.
Dilute and store
Dilute cDNA 1:5–1:10 with nuclease-free water for qPCR; store at −20 °C.
Critical points
- Use the same RNA input and the same kit for all samples you compare.
- No-RT controls reveal gDNA contamination.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Late Ct values | Increase RNA input, check RNA integrity, use a mix of oligo(dT) and random primers. |
| Signal in no-RT | Repeat DNase treatment; design intron-spanning primers. |
Safety
Standard laboratory care.
Further reading
- Manufacturer instructions for your reverse transcription kit.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.