<P> Two common methods for the detection of PCR products in real - time PCR are: (1) non-specific fluorescent dyes that intercalate with any double - stranded DNA, and (2) sequence - specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence . </P> <P> The Minimum Information for Publication of Quantitative Real - Time PCR Experiments (MIQE) guidelines propose that the abbreviation qPCR be used for quantitative real - time PCR and that RT - qPCR be used for reverse transcription--qPCR . The acronym "RT - PCR" commonly denotes reverse transcription polymerase chain reaction and not real - time PCR, but not all authors adhere to this convention . </P> <P> Cells in all organisms regulate gene expression by turnover of gene transcripts (single stranded RNA): The amount of an expressed gene in a cell can be measured by the number of copies of an RNA transcript of that gene present in a sample . In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary . The polymerase chain reaction (PCR) is a common method for amplifying DNA; for RNA - based PCR the RNA sample is first reverse - transcribed to complementary DNA (cDNA) with reverse transcriptase . </P> <P> In order to amplify small amounts of DNA, the same methodology is used as in conventional PCR using a DNA template, at least one pair of specific primers, deoxyribonucleotides, a suitable buffer solution and a thermo - stable DNA polymerase . A substance marked with a fluorophore is added to this mixture in a thermal cycler that contains sensors for measuring the fluorescence of the fluorophore after it has been excited at the required wavelength allowing the generation rate to be measured for one or more specific products . This allows the rate of generation of the amplified product to be measured at each PCR cycle . The data thus generated can be analysed by computer software to calculate relative gene expression (or mRNA copy number) in several samples . 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 . This measurement is made after each amplification cycle, and this is the reason why this method is called real time PCR (that is, immediate or simultaneous PCR). In the case of RNA quantitation, the template is complementary DNA (cDNA), which is obtained by reverse transcription of ribonucleic acid (RNA). In this instance the technique used is quantitative RT - PCR or Q - RT - PCR . </P>

Which of the following is not an example of an amplification method