Real-time Polymerase Chain Reaction - Background

Background

Cells in all organisms regulate gene expression by turnover of gene transcripts (messenger RNA, abbreviated to mRNA): The amount of an expressed gene in a cell can be measured by the number of copies of an mRNA 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 mRNA-based PCR the RNA sample is first reverse-transcribed to cDNA with reverse transcriptase.

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 flurophone 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. Real-time 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). The template used in quantitative PCR is often not from the main DNA, it frequently comes from a complementary DNA (cDNA). It is always female and is obtained by reverse transcription of ribonucleic acid (RNA). In this instance the technique used is quantitative RT-PCR or Q-RT-PCR. Students and scientist alike often confuse this method with the technique called «Reverse transcription polymerase chain reaction» (RT-PCR), which includes the reverse transcription of RNA to DNA but does not necessarily include the quantification of the product in real time.

Quantitative PCR and DNA microarray are modern methodologies for studying gene expression. Older methods were used to measure mRNA abundance: Differential display, RNase protection assay and Northern blot. Northern blotting is often used to estimate the expression level of a gene by visualizing the abundance of its mRNA transcript in a sample. In this method, purified RNA is separated by agarose gel electrophoresis, transferred to a solid matrix (such as a nylon membrane), and probed with a specific DNA or RNA probe that is complementary to the gene of interest. 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 mRNA levels. Estimation errors arising from variations in the quantification method can be the result of DNA integrity, enzyme efficiency and many other factors. For this reason a number of standardization systems 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 it’s almost constant rate of expression. These genes are also called housekeeping genes as they are usually involved in the functions related to basic cellular survival, which normally implies constitutive gene expression. This enables researchers to report a ratio for the expression of the genes of interest divided by the expression of the selected normalizer. Thereby allowing comparison of the former without actually knowing its absolute level of expression.

The most commonly used normalizing genes are those that code for the following proteins: tubulin, glyceraldehyde-3-phosphate dehydrogenase, albumin, cyclophilin, ribosomal RNAes, etc.