What is Real Time PCR & RT-QPCR

The difference between quantitative real-time PCR (qPCR) and end-point PCR is the step at which the PCR product amount is measured. In traditional PCR, product quantitation is performed once at the end of the reaction. In qPCR, the amount of PCR product is measured after every amplification round. qPCR instruments are designed for both PCR cycling and the detection of signal from fluorescent dyes during amplification. By monitoring the progress of amplification in real time, the point in time when the amplification of the target is detected above background can be identified. The fewer targets present in the reaction, the later the first significant signal is detected. This technique, therefore, is of great advantage when gene-expression differences are analyzed.

There are two ways to detect PCR signal with the help of fluorescent dyes: fluorescent dyes that nonspecifically bind to double-stranded DNA (Dye-based qPCR) or fluorescently labeled, sequence-specific probes (Probe-based qPCR).

In Dye-based qPCR  the use of universal fluorescent DNA-intercataling dyes enables analysis of many targets without having to synthesize sequence-specific probes. However, this method has the disadvantage of detecting all specific and unspecific amplification events as well as primer–dimers because the dye binds to all double-stranded DNA. To achieving high PCR specificity, avoiding background and primer–dimer formation is critical.

Probe-based qPCR method is more precise, as the use of sequence-specific fluorescently labeled probes ensures the detection of the target product only. A variety of probe fluorescence chemistry is currently available. Multiple fluorescent probes can be used in multiplex qPCR, although the use of specific probes is more expensive than using DNA-intercalating dyes.

qRT-PCR One Step Kits allow reverse transcription and real-time PCR to be performed in one step. In this case, RNA is the template for qPCR. One-step qPCR kits include separate qPCR and reverse transcription master mixes. Same like qPCR, the qRT-PCR One Step Kits are also available in both Dye-based and Probe-based formats.

Many real-time PCR instruments available on the market require a passive internal reference dye (either ROX™ or fluorescein) for normalization of the fluorescent signal. The use of passive reference dyes does not interfere with the qPCR. The reference dyes are either supplied separately, or they can be included in the qPCR mastermix at appropriate concentration. biotechrabbit offers both. The choice depends on your instrument requirements for reference dye.