Validating quantitative data model
It monitors the amplification of a targeted DNA molecule during the PCR, i.e.
For this reason a number of standardization systems (often called normalization methods) have been developed.
Some have been developed for quantifying total gene expression, but the most common are aimed at quantifying the specific gene being studied in relation to another gene called a normalizing gene, which is selected for its almost constant level of expression.
In addition, in four step PCR the fluorescence is measured during short temperature phase lasting only a few seconds in each cycle, with a temperature of, for example, 80 °C, in order to reduce the signal caused by the presence of primer dimers when a non-specific dye is used.
The temperatures and the timings used for each cycle depend on a wide variety of parameters, such as: the enzyme used to synthesize the DNA, the concentration of divalent ions and deoxyribonucleotides (d NTPs) in the reaction and the bonding temperature of the primers.
The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines propose that the abbreviation q PCR be used for quantitative real-time PCR and that RT-q PCR be used for reverse transcription–q PCR.
Quantitative PCR can also be applied to the detection and quantification of DNA in samples to determine the presence and abundance of a particular DNA sequence in these samples.
Older methods were used to measure m RNA abundance: Differential display, RNase protection assay and Northern blot.The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase.The PCR process generally consists of a series of temperature changes that are repeated 25 – 50 times.In the case of RNA quantitation, the template is complementary DNA (c DNA), which is obtained by reverse transcription of ribonucleic acid (RNA).In this instance the technique used is quantitative RT-PCR or Q-RT-PCR.A real-time polymerase chain reaction (Real-Time PCR), also known as quantitative polymerase chain reaction (q PCR), is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).