Real-Time PCR, also referred to as Quantitative PCR (or qPCR), was developed as a precise, efficient and rapid method for nucleic acid detection.
This technique is based on traditional Polymerase Chain Reaction (PCR) technology with a few improvements:
- Real-Time PCR combines nucleic acid amplification and detection in one step
- It can use smaller amounts of starting material than traditional PCR
- It is possible to quantify the product based on fluorescent detection
- There is no post-amplification process
The detection of template DNA (or RNA if a reverse transcription step is added prior to amplification) is live and based on when the PCR product is amplified above the threshold of background or the cycle threshold number (Ct number).
Far different from the former end-point method of standard PCR techniques, real-time PCR can be quantitative because the PCR product is detected using fluorescent dyes in real time.
Thus, real-time PCR has earned the alternate and distinctive name of quantitative PCR.
Key Applications of Real-Time PCR:
While qPCR has been used for a wide variety of applications over the years, several uses of technique continue to be particularly popular.
These include:
- Gene expression (mRNA) analysis
- microRNA and non-coding RNA analysis
- Genetic variation analysis
- Mutation detection
- SNP analysis
- Genotyping/allelic discrimination
- Detection of pathogens and infections
Real-Time Instruments:
When it comes to instrumentation, purchasing a Real-Time Thermocycler can be a significant investment.
Some things to consider before purchasing a qPCR machine include the overall footprint, the block formats available, the run times, expandability, the software used, volume limits for wells, etc.
You can compare specifications on some of the latest instruments available in the
qPCR Machines Search Tool
, including some examples shown below:
Range of Real-Time PCR Kits and Reagents:
As with most techniques there are now a number of ready-made kits available to make qPCR easier and more reproducible.
qPCR Kits
can be purchased which include all reagents needed for a successful reaction. These kits usually include everything you need to run a qPCR reaction except the primers for your particular gene of interest.
Individual components can also be purchased; this includes everything from
qPCR master mixes,
qPCR assays and other
consumables.