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Validating quantitative data model

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.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.Quantitative PCR and DNA microarray are modern methodologies for studying gene expression.Older methods were used to measure m RNA abundance: Differential display, RNase protection assay and Northern blot.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).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 m RNA copy number) in several samples.

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 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 real-time, and not at its end, as in conventional PCR.Real-time PCR can be used quantitatively (quantitative real-time PCR), and semi-quantitatively, i.e.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 (c DNA) with reverse transcriptase.Real-time PCR technique can be classified by the chemistry used to detect the PCR product, specific or non-specific fluorochromes.A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye.Although this technique is still used to assess gene expression, it requires relatively large amounts of RNA and provides only qualitative or semi quantitative information of m RNA levels.Estimation errors arising from variations in the quantification method can be the result of DNA integrity, enzyme efficiency and many other factors.


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