RT-qPCR
Reverse transcription is run as a separate reaction, usually primed with oligo(dT) or random hexamers, and the resulting cDNA is diluted and stored so that any number of assays can be run from it. This is the flexible option and the one most expression work uses: the cDNA can be re-run, re-assayed and diluted until carry-over inhibition no longer matters.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Two-step
Reverse transcription is run as a separate reaction, usually primed with oligo(dT) or random hexamers, and the resulting cDNA is diluted and stored so that any number of assays can be run from it. This is the flexible option and the one most expression work uses: the cDNA can be re-run, re-assayed and diluted until carry-over inhibition no longer matters.
How it differs from the standard method
- One cDNA stock serves every assay, so several genes can be measured from one aliquot of RNA.
- The cDNA can be diluted, which is the simplest and often the only remedy for inhibitors carried out of the extraction.
- A failed plate can be re-run from the same cDNA, and a question thought of later can still be asked.
- The extra open-tube step adds a contamination route and an RT batch effect: samples reverse transcribed on different days with different mixes carry an offset that is not biology.
- Freeze–thaw of the cDNA stock drifts, so single-use aliquots are worth the plastic.
1Two enzymes in series, one number at the end
RT-qPCR is not a technique in its own right so much as a pair of them bolted together: a reverse transcription that turns RNA into cDNA, and a quantitative PCR that counts the cDNA. The qPCR half is well behaved and heavily instrumented: you watch every cycle of it happen, you can measure its efficiency on a dilution series, and a melt curve or a probe tells you what it amplified.
Reverse transcription
Off the plate, no readout, efficiency between a few per cent and a few tens of per cent, and it varies between samples. Its one saving grace: a given reaction's efficiency multiplies every transcript in that sample by the same factor.
Quantitative PCR
On the plate, one reading per cycle, efficiency measurable from a standard curve, specificity checkable with a melt curve or a probe. Contributes little variance when it is set up properly.
Normalisation
Measuring a reference gene in the same well divides out anything that scaled the whole sample: input mass, RT efficiency, inhibition. It is why relative quantification is robust in spite of step one.
A reference that moves
If the normaliser responds to your treatment, its change is subtracted from your answer. No amount of replication finds this; only testing the reference in your own condition does.
2Why normalisation works, and exactly where it stops
A per-sample reverse-transcription efficiency is a multiplicative factor on every transcript in that sample. So is the amount of RNA you put in, and so, to a first approximation, is an inhibitor that slows the polymerase in that well.
3One tube or two
In a two-step workflow the reverse transcription is a separate reaction: you make a cDNA stock from each RNA sample, dilute it, and run as many assays off it as you like, now or in six months. In a one-step reaction the RNA goes into a sealed tube containing both enzymes, and the instrument runs fifteen minutes at 50 °C before starting to cycle. The two differ in flexibility and in reproducibility, and they differ in opposite directions.
- Two-step
- Reverse transcribe in bulk with oligo(dT) or random hexamers, then run any assay on the cDNA. Flexible, re-runnable, dilutable; one extra open-tube handling step per sample and an RT whose efficiency varies between batches.
- One-step
- RT and PCR in one sealed tube, primed by the assay's own reverse primer. Fewer pipetting steps, less contamination risk, tighter replicates; one assay per tube, the RNA prep goes in undiluted, and the cDNA is gone when the run ends.
- ΔCq
- Target Cq minus reference Cq within one sample. This is where the cancellation happens, so both must come from the same cDNA and the same run.
- ΔΔCq
- The difference between two samples' ΔCq values; 2^−ΔΔCq is the fold change. The 2 is an assumption about efficiency, not a constant of nature.
- Reference gene
- A transcript asserted not to change under your treatment. An assertion to be tested in your own system, ideally two or three of them combined as a geometric mean.
- Minus-RT control
- The RNA, the mix, no reverse transcriptase. Catches genomic DNA in the sample. In a one-step assay it needs its own master mix, which is why it gets skipped.
- No-template control
- Water in place of sample. Catches template or amplicon in the reagents. It does not catch genomic DNA in your RNA, which is the other control's job.
- RT replicate
- An independent reverse transcription of the same RNA, as opposed to another well off the same cDNA. The only kind of replicate whose spread includes the first enzymatic step.
4The controls, and what each of them actually rules out
Two blanks do two different jobs and are not interchangeable. The no-template control is the mix with water instead of sample: if it amplifies, there is template or previous amplicon in your reagents, and the run is void.
Key terms
Practise these as flashcards →The fractional cycle at which a well crosses the quantification threshold. A cycle number, not an amount; it becomes an amount only because the reaction doubles.
The fraction of RNA copies converted to cDNA. Unmeasured in a normal experiment, variable between samples, and multiplicative across every transcript in a given reaction.
Averaging several reference genes' Cq values (equivalent to a geometric mean of their copy numbers) to normalise, which also reveals whether any one of them is moving.
RT buffer, residual primer and extraction residues slowing the polymerase. Shows as late Cq values across the plate and is diluted away, not washed away.
An amplicon whose primers sit in different exons, so genomic DNA cannot be amplified across it. Design-level protection against DNA contamination.
Several wells of one cDNA versus independent samples each taken through extraction and RT. Only the second supports a biological claim.
The reporting guidelines for quantitative PCR experiments: which controls, efficiencies, normalisers and replicate structures have to be stated in a paper.
Replacing the 2 in 2^−ΔΔCq with each assay's measured (1 + E). Needed when target and reference efficiencies differ, and harmless when they do not.
When to use it
- Measuring how much a known transcript changes between conditions, which is the single most common quantitative question asked of RNA.
- Validating a handful of genes picked out of an RNA-seq or microarray experiment, on more samples than sequencing them again would be worth.
- Measuring a transcript at low abundance in a small sample, where the dynamic range and sensitivity of PCR beat hybridisation and sequencing on cost.
- Clinical and diagnostic detection of RNA viruses, where a one-step closed-tube reaction with an internal control is the standard format.
- Any time you need absolute copy numbers of a transcript, with a standard curve and the caveat that the RT step's efficiency is not captured by a DNA standard.
Compared with alternatives
The second half of this workflow, and where the standard curve, the amplification efficiency and the melt curve belong. Read that page for how a Cq is defined and how to measure efficiency; this one is about what the reverse transcription adds.
The first half. Priming strategy, input matching and the minus-RT control are decided there, and they set the ceiling on what the qPCR can report.
Partitions the same cDNA and counts positive partitions, so it gives absolute copies without a standard curve and tolerates inhibitors better. Prefer it for rare targets, copy-number work and when there is no good reference gene.
Measures everything rather than what you chose to ask about, and needs no reference gene. Prefer it for discovery; RT-qPCR remains cheaper and more precise for a few genes across many samples.
Reports transcript size and isoform state, which no PCR readout can. Worth running once when an assay's amplicon could be sitting on more than one isoform.
The older way of measuring many transcripts at once; RT-qPCR was the standard method for confirming array hits and is still used the same way for sequencing hits.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Reverse transcription is run as a separate reaction, usually primed with oligo(dT) or random hexamers, and the resulting cDNA is diluted and stored so that any number of assays can be run from it. This is the flexible option and the one most expression work uses: the cDNA can be re-run, re-assayed and diluted until carry-over inhibition no longer matters.
- One cDNA stock serves every assay, so several genes can be measured from one aliquot of RNA.
- The cDNA can be diluted, which is the simplest and often the only remedy for inhibitors carried out of the extraction.
- A failed plate can be re-run from the same cDNA, and a question thought of later can still be asked.
- The extra open-tube step adds a contamination route and an RT batch effect: samples reverse transcribed on different days with different mixes carry an offset that is not biology.
- Freeze–thaw of the cDNA stock drifts, so single-use aliquots are worth the plastic.