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Short answer
Reverse transcription PCR (also written reverse-transcriptase PCR, RT-PCR) turns RNA into complementary DNA (cDNA) with a reverse transcriptase enzyme and then amplifies that cDNA by PCR. It can be run in one tube (one-step) or in two separate reactions (two-step), and the choice of RT primer changes sensitivity, so both belong in the methods you report.
What reverse transcription PCR is
PCR amplifies DNA. To measure or detect an RNA molecule, the RNA must first be copied into DNA: Roche describes this as a reverse transcriptase enzyme mediating the creation of a DNA complement from RNA. The cDNA then serves as the template for the PCR step. The method is also called reverse-transcriptase PCR or reverse transcription PCR; these are the same technique under different spellings.
In this guide “RT” always means reverse transcription. The abbreviation is sometimes confused with real-time PCR; the quantitative variant is covered in qPCR and RT-qPCR: how they differ, and the reaction mechanics shared with ordinary PCR are in PCR protocol basics.
What is reverse transcription PCR?
It is a two-stage workflow: reverse transcribe RNA into cDNA, then amplify the cDNA by PCR. Both stages can share one tube or be run separately, which defines the two main formats below.
What is reverse transcriptase PCR?
It is the same method. “Reverse transcriptase” names the enzyme that performs the first stage; “reverse transcription” names the reaction. Either phrase refers to RNA-to-cDNA conversion followed by PCR.
One-step versus two-step
Roche defines the formats by tube: if the RT step is performed in the same tube as the PCR, the process is called one-step; when RT and PCR are performed in separate tubes, it is called two-step. Wacker and Godard (2005) describe the two-step method as creating cDNA first in a separate reverse transcription reaction.
| Feature | One-step | Two-step | Source |
|---|---|---|---|
| Reaction layout | RT and PCR combined in a single reaction | RNA reverse-transcribed first; an aliquot goes to the PCR | QIAGEN |
| Primers | Requires gene-specific primers | Oligo-dT, random or gene-specific RT primers can be used | QIAGEN |
| Handling | Easy handling, fast, reproducible, low contamination risk; quicker to set up and less expensive to use | Multiple PCRs can be run from one RT reaction | QIAGEN; Wacker and Godard |
| Leftover material | Other genes of interest cannot be amplified later for analysis | cDNA can be stored long term | QIAGEN; Wacker and Godard |
The practical consequence is the cDNA archive. A two-step workflow leaves a stored cDNA pool that can be re-used for further targets; a one-step workflow uses gene-specific primers in a single reaction, so other genes cannot be amplified from it later and the original RNA must be aliquoted for further testing (Wacker and Godard).
Choosing the RT primer
QIAGEN lists three RT primer types: oligo-dT primers (typically 13-18mers), random oligomers (such as hexamers, octamers or nonamers), and gene-specific primers. Roche adds the usual rationale for each.
- Oligo(dT): used for full-length copies of mRNA (Roche).
- Random hexamers: used when the RNA is long or lacks a poly(A) tail, for example prokaryotic mRNA (Roche).
- Gene-specific primers: enhance sensitivity by directing all of the RT activity to a specific message; in one-step formats the RT primer doubles as the reverse PCR primer (Roche).
Priming is not a neutral detail. Wacker and Godard report that different priming methods in the RT step provide different sensitivities and efficiencies, and that in their experiments a one-step method with gene-specific priming may be more sensitive for quantifying certain genes such as PolR2A (a lower-expressed mRNA, with a 5-cycle lower threshold). That is a finding about specific genes and a specific enzyme system, not a general rule.
Keeping genomic DNA out
Because the PCR step amplifies DNA, contaminating genomic DNA can be amplified alongside cDNA. QIAGEN’s guidance is to design primers so that one half anneals to one exon and the other half to the adjacent exon, or so that the primers flank an intron, which makes the genomic product larger than the cDNA product, and to use DNA-free RNA. Sample preparation is covered in RNA extraction protocol basics.
Where it matters for labs and supply decisions
The sourced points above turn into a short checklist for planning an assay and its reagent needs.
| Decision | What the sources say | Consequence |
|---|---|---|
| One-step or two-step | One-step needs gene-specific primers and leaves nothing for other genes later (QIAGEN; Wacker and Godard) | Reagent lists differ: a one-step design needs a combined RT-PCR reaction, a two-step design needs separate RT and PCR reagents and storage for the cDNA |
| RT primer type | Oligo-dT, random and gene-specific primers serve different RNA types and goals (QIAGEN; Roche) | Primer choice should follow the RNA type (for example poly(A)-lacking RNA points to random primers) and be recorded |
| RNA input | The total amount of RNA in the reaction can affect RT efficiency (Wacker and Godard) | Keep RNA input consistent across samples that will be compared |
| Genomic DNA | Intron-spanning or exon-junction primers and DNA-free RNA (QIAGEN) | Check primer design before ordering oligonucleotides |
| RT enzyme | Roche notes that endogenous RNase H activity in some reverse transcriptases degrades the RNA of an RNA:DNA hybrid, which matters for cDNA quality in the PCR step | Enzyme properties differ by product; check the enzyme’s own documentation |
For how RT-PCR results are reported and quantified, see MIQE guidelines: the qPCR reporting checklist and Ct values in qPCR.
Common misunderstandings
- “RT-PCR is the same as qPCR.” No. RT refers to the reverse transcription step; quantitative PCR is a separate readout. The relationship is explained in the RT-qPCR guide.
- “One-step is simply the better version.” The sources describe trade-offs, not a winner: one-step is faster and lower in contamination risk, two-step keeps cDNA for other targets.
- “Any RT primer gives the same result.” Wacker and Godard report different sensitivities and efficiencies with different priming methods.
- “RNA is all that is amplified.” The PCR step amplifies DNA, which is why genomic DNA control matters.
FAQ
What is reverse transcription PCR?
A method that converts RNA into cDNA with reverse transcriptase and then amplifies the cDNA by PCR, in one tube (one-step) or two separate reactions (two-step).
What is reverse transcriptase PCR?
The same method, named after the enzyme that performs the RNA-to-cDNA step. Reverse transcriptase PCR, reverse-transcriptase PCR and reverse transcription PCR are interchangeable spellings.
Which RT primer should be used?
It depends on the RNA and the goal: oligo(dT) for full-length mRNA copies, random primers for long or non-polyadenylated RNA, gene-specific primers for sensitivity to one target (Roche; QIAGEN).
Sources
- Wacker MJ, Godard MP. “Analysis of One-Step and Two-Step Real-Time RT-PCR Using SuperScript III.” J Biomol Tech 2005;16(3):266-271. https://pmc.ncbi.nlm.nih.gov/articles/PMC2291734 (checked 2026-10-29).
- QIAGEN, “Guidelines for RT-PCR” bench guide. https://www.qiagen.com/ma/knowledge-and-support/knowledge-hub/bench-guide/pcr/real-time-rt-pcr/guidelines-for-rt-pcr (checked 2026-10-29).
- Roche Life Science, “Reverse transcription technology.” https://lifescience.roche.com/global_en/articles/reverse-transcription-technology.html (checked 2026-10-29).
