Biological

RT-PCR

Real Time PCR

Real-time PCR amplifies a chosen DNA or RNA target and reads its fluorescence after every cycle. It quantifies gene expression, pathogen load or copy number from very small amounts of material.

1 test optionFluorescence read every cycleRelative or absolute quantificationTypical turnaround 5–7 working days
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RT-PCR measurement principleIllustrative
Excitation lightDetectorPCR tubesHeated blockThresholdAmplification curve
Copies counted as they are madeIllustrative
01 / Overview

What is RT-PCR?

A target sequence is copied by repeated heating and cooling cycles while a fluorescent dye or probe reports the amount of product after each cycle. The cycle at which the signal crosses a threshold (Ct) comes earlier when there is more target to start with. RNA is first reverse-transcribed into cDNA.

Unlike end-point PCR read on a gel, real-time PCR measures during amplification, so it is quantitative over a wide range and needs no handling after the run. Reference genes, standards and controls let results be reported as fold changes or copy numbers.

02 / How it works

How it works

  1. 01

    Template is prepared

    DNA or RNA is extracted; RNA is reverse-transcribed into cDNA for gene-expression work.

  2. 02

    Reaction is set up

    Template, primers and a fluorescent dye or probe are mixed and loaded into plate wells.

  3. 03

    Cycles are read

    The block heats and cools 35–45 times while fluorescence is measured in every well.

  4. 04

    Ct values are analysed

    The cycle where the signal crosses a threshold gives Ct, used for fold change or copy number.

03 / What it measures

What it measures

Gene expression

Compare mRNA levels between samples as fold change against reference genes.

Useful forTreatment studies, cell lines, tissues

Pathogen & target detection

Detect specific DNA or RNA sequences, such as microbes or GM elements.

Useful forMicrobial, food and plant samples

Absolute quantification

Count copies of a target against a standard curve of known amounts.

Useful forViral load, copy number, vector titre

Genotyping & SNPs

Call alleles with probe-based allelic discrimination.

Useful forPlant and animal lines, breeding panels

Melt-curve specificity

Check that a single, specific product was amplified.

Useful forDye-based assays, primer validation
04 / Test options

Choose the RT-PCR options you need

1 option · none added yet

RT-PCR test options
05 / Sample requirements

Sample requirements

Accepted forms
Purified RNA, DNA or cDNA; cells or tissue for extraction on request
Quantity
Around 1–2 µg RNA, or 20–50 µL DNA/cDNA per sample
Purity
A260/A280 around 1.8–2.0, free of inhibitors
Primers & controls
Primer or probe sequences, reference genes and controls
Transport
RNA frozen on dry ice; DNA chilled or frozen
  • Use nuclease-free, sealed and clearly labelled tubes
  • Share primer sequences, reference genes and product size
  • Include untreated or calibrator samples where relevant
  • No repeatedly freeze–thawed RNA
  • No live infectious or unprocessed high-risk samples

Hazardous or air-sensitive samples: mention it in your request, and attach the MSDS if you have one.

06 / Results & turnaround

Results & turnaround

What you receive

  • Amplification plots for every well
  • Ct values with replicate statistics
  • Melt curves for dye-based assays
  • Fold change (ΔΔCt) or copy numbers
  • Raw run files
  • Report PDF

Turnaround & pricing

Typically 5–7 working days after samples reach the lab.

Price confirmed in your quotation, depending on user type, options and number of samples.

Sample report · IllustrativeExample Ct value24.6Illustrative — not measured sample data
07 / Limitations

When RT-PCR isn't the right fit

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