Real-Time PCR Thermal Cyclers/ Thermo cyclers, also referred to as Quantitative PCR (qPCR) calculate PCR amplification as it occurs. Using smaller amounts of starting material and combining nucleic acid amplification and detection allows for efficiency and eliminates the post-amplification process. Real-time PCR thermo cyclers can be used with both DNA and RNA.Synthesis of cDNA from total RNA samples is the first step in the two-step RT-PCR gene expression quantification experiment. Real-time or Quantitative PCR and RT-PCR use the linearity of DNA amplification to determine absolute or relative amounts of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation.In quantitative PCR, DNA amplification is monitored at each cycle of PCR. When the DNA is in the log linear phase of amplification, the amount of fluorescence increases above the background. The point at which the fluorescence becomes measurable is called the Quantification Cycle (Cq) or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against Cq. The amount of DNA or cDNA in an unknown sample can then be calculated from its Cq value.