qPCR – one of the most powerful and sensitive gene analysis techniques available
The Quantitative Polymerase Chain Reaction (qPCR) is one of the most powerful and sensitive gene analysis techniques available and is used extensively in industrial, academic and diagnostic labs.
Historically, quantitative analysis of DNA/RNA has relied on Southern/ Northern blot hybridisation or conventional end-point PCR. To perform these techniques, a sample of unknown concentration is estimated by comparing the intensity of the amplified band on a gel or blot after a PCR reaction to standards of a known concentration. However there are many limitations to these methods which qPCR has overcome.
The qPCR reaction is performed on a thermal cycling instrument that uses fluorescence detection chemistry to monitor the amount of DNA present in each cycle of the PCR reaction. This is often referred to as Real-Time analysis. As the amount of target DNA in the reaction increases, so will the amount of fluorescence emitted from the marker. By comparing the normalised fluorescence of known standards against the PCR cycle number, the amount of DNA in an unknown sample can be accurately determined.
One-Step RT-PCR can also be performed in real time (qRT-PCR). This technique offers the most sensitive and convenient method of quantifying gene expression.
The Advantages of qPCR
• Time Saving: No need to run a gel. The data is quantified by the instrument as the PCR reaction progresses.
• Increased Sensitivity: Can detect low abundance templates that would not normally be visualised by conventional gel-based methods.
• Increased Accuracy: Readings are taken during exponential phases of the PCR reaction. Therefore there will be no bias from limiting reagent concentrations (figure 1.3.1).
• Reduced Risk of Contamination:The closed tube format reduces the exposure of the reaction to contaminants.
ABgene® have developed a versatile range of ABsolute™ qPCR Master Mixes optimised to give sensitive and reproducible results every time. All ABgene® ABsolute™ qPCR reagents are in a convenient master mix format to minimise the number of reagent handling steps saving time and ensuring reproducibility. ABsolute™ qPCR Mixes contain Thermo-Start® DNA Polymerase. This proprietary enzyme is a chemically modified patented version of Thermoprime Plus DNA Polymerase that is inactive until ‘switched on’ by high temperature incubation (hot-start), eliminating non-specific priming so that only the target DNA is amplified. The ABsolute™ qRT-PCR Mixes also contain ABgene®’s unique Reverse-iT™ RTase Blend with RNase inhibitors to overcome problematic RNA templates and increase sensitivity. All kits are supplied in a reaction buffer optimised to give maximal performance for each application.
ROX (optional with all ABsolute™ qPCR Mixes) is used as an internal reference by some sequence detection instruments to normalise fluorescence. ROX reference dye is available included in the mix or as a separate vial for optimisation.
ABsolute™ qPCR Mixes are optimised for use with TaqMan® probes, Molecular Beacons and other popular detection chemistries and have been extensively tested with most 96- and 384-well Real-Time PCR instruments including the ABI PRISM® range, Stratagene Mx4000™ and Bio-Rad iCycler IQ™.