End-point PCR tells you whether a sequence is there, qPCR tells you how much, and RT-qPCR does the same for RNA. Here is what changes between them and the reagent list for each.
Range: qPCR and RT-PCR reagents4 minute readUpdated
The polymerase chain reaction copies a stretch of DNA between two primers. Each cycle of denaturation, annealing and extension roughly doubles the target, so after 30–35 cycles a single sequence becomes billions of copies. You see the result at the end, usually as a band on an agarose gel sized against a DNA ladder.
Because the reaction plateaus, band brightness at the end says little about how much template you started with. End-point PCR answers yes-or-no questions: genotyping, colony screening, checking for a contaminant, or making a fragment to clone.
qPCR measures fluorescence after every cycle. Either a dye that binds double-stranded DNA (SYBR Green type) or a sequence-specific probe reports the product as it accumulates. The cycle at which the signal crosses a threshold, the quantification cycle or Cq, falls by about one for every doubling of starting template. Compared against a standard curve or a reference gene, Cq gives absolute or relative quantity.
Dye-based qPCR is followed by a melt curve, which confirms that a single product was made. Probe-based qPCR adds specificity and lets you measure several targets in one well.
Polymerases that copy DNA cannot use RNA as a template. Reverse transcriptase copies RNA into complementary DNA (cDNA) first, and the cDNA is then amplified. With end-point PCR this is RT-PCR; with real-time detection it is RT-qPCR, the standard method for measuring gene expression and RNA viruses.
| One-step | Two-step | |
|---|---|---|
| Setup | Reverse transcription and qPCR in one tube | cDNA made first, then used in separate qPCR reactions |
| Primers for RT | Gene-specific | Oligo(dT), random primers or a mix |
| Strengths | Fast, less handling, lower contamination risk | One cDNA stock for many genes; cDNA can be stored |
| Best for | Many samples, few targets | Few samples, many targets |
| Method | Core reagents |
|---|---|
| End-point PCR | Taq or a PCR premix (polymerase, buffer, Mg²⁺, dNTPs), primers, template, a DNA ladder for the gel |
| qPCR | qPCR premix with dye or for probes, primers (and probe), template, ROX if your instrument needs it |
| Two-step RT-qPCR | Reverse transcription kit (reverse transcriptase, buffer, dNTPs, primers, RNase inhibitor), then the qPCR reagents |
| One-step RT-qPCR | One-step mix combining reverse transcriptase and hot-start polymerase, gene-specific primers |
All of these are in the qPCR and RT-PCR range. The master mix guide explains how to match a premix to your chemistry and instrument.
RT-qPCR can only be as good as the RNA going into it.
| Control | Detects | Needed for |
|---|---|---|
| No-template control | Contamination, primer-dimers | All methods |
| No-RT control | Signal from genomic DNA | RT-PCR and RT-qPCR |
| Positive control | Failed reagents or cycling | All methods |
| Reference genes | Differences in input and RT efficiency | Relative RT-qPCR |
PCR shows the product at the end of the reaction, usually on a gel, which tells you whether a sequence is present and its size. qPCR measures the product during every cycle with a fluorescent dye or probe, which tells you how much template you started with.
No. RT-PCR includes reverse transcription of RNA into cDNA before amplification; qPCR is real-time quantitative PCR. When both are combined to quantify RNA, the method is called RT-qPCR.
Use one-step when you test many samples for a few targets and want speed and less handling. Use two-step when you want to test many genes from the same samples, or keep cDNA for later experiments.
It shows whether any signal comes from contaminating genomic DNA rather than RNA. If the no-RT control amplifies, add a DNase step or redesign primers to span an exon-exon junction.
For research use only. Protocols here are general outlines: follow your kit manual and your institution’s safety rules. See our editorial policy.
Products for this workflow
Taq DNA polymerase, PCR and qPCR premixes, and reverse transcription kits for cDNA synthesis.
qPCR / RT-PCR
A 2X qPCR master mix looks like a commodity until it does not match your instrument or chemistry. This guide covers dye vs probe, ROX levels, hot-start, setup volumes and how to validate efficiency.
4 minute read
DNA and RNA extraction
Every extraction does the same four things: lyse, separate, wash, elute. The chemistry you use for each step decides yield, purity and how many samples you can run a day.
4 minute read
qPCR / RT-PCR
Your controls usually say what went wrong. A positive control, a no-template control, a no-RT control and one dilution of the sample separate template, inhibition and primer problems.
3 minute read
Send the product names or catalogue numbers, quantities and delivery country. We'll confirm availability, lead time and the documents for qpcr and rt-pcr reagents.