PCR/Amplification

Quantitative PCR

  What is real time PCR (qPCR)?

Quantitative PCR (qPCR) or Real-Time PCR uses fluorescent reporter molecules to allow quantification of amplified products. As in conventional PCR, a DNA, cDNA, or RNA template is amplified, but at each cycle, fluorescence is monitored for relative or absolute quantification. This technique is useful for numerous areas of research including gene expression analysis, genotyping, microRNA analysis, genetic variation analysis, and protein analysis.


  How to analyze qPCR data:

The fluorescence of the reporter molecules is measured and quantified; typically these are either dyes (i.e. SYBR Green, Ethidium Bromide) that intercalate – bind between – the bases in double-stranded DNA, or probes designed to bind a specific sequence (i.e. Molecular Beacons, TaqMan® probes) on the DNA.

There are two major quantification methods for qPCR data. Relative quantification, the more common method, uses ΔΔCT information, by which the expression or abundance ratio of the target gene in the sample is determined, compared to a control gene, and normalized with the expression ratio of a reference gene. Since, the efficiency E values for target and reference genes differ, this method also accounts for the differences in E values. This method is more frequently employed in gene expression analysis.

The second method, absolute quantification, is more common in environmental microbiology and makes use of the standard-curve (SC). The SC method uses dilution series of known template concentration, N0, to create a standard curve using the linear regression of log(N0) versus the CT (threshold cycle). This is then used to calculate template concentrations of the sample. The basis of this method is that the efficiency value of the sample is the same as that of the standard.1,2

Sigma has developed various qPCR kits, either with probes or SYBR® Green to help you get started or improve your qPCR results. All of our products are prepared as ReadyMixes for your convenience; explore our KiCqStart® for the easiest, fastest, most convenient qPCR options available.

Not sure what product suits you best? Check out our PCR Selection Guide.
 

Routine PCR  |  RT-PCR  |  PCR Labware  |  dNTPs

 

SYBR Green
Our SYBR Green product lines offer a wide variety of options to meet your qPCR needs. These include:
• Hot-Start
• Instrument Optimized or Optimizable
• Fast or Traditional

And all our products are available as convenient, ready-to-use ReadyMixes.

 
Probe-Based
Our Probe-based products offer a variety of features to help meet your experimental needs.
These include:
• Hot-Start
• Multiplex Options
• Fast or Traditional

And all our products are available as convenient, ready-to-use ReadyMixes.
 
Quantitative RT-PCR
Explore our Quantitative RT-PCR options!! We offer various types of products for convenience and quality:
• JumpStart RT-qPCR
KiCqStart One-Step Probe RT-qPCR
MystiCq® MicroRNA RT-qPCR

Explore our page to learn more.


 
PCR Selection Guide
Choose the perfect PCR product for your experiment.

Use our Selection Guide to find which of our qPCR products best suits your experimental capacity and needs. Choose by SYBR Green/probe-based, by instrument, or both.
 
Custom Products
• Predesigned KiCqStart Primers
• Custom qPCR Probes
• Custom Oligonucleotides