Molecular strategies for the studies of the expression of gene variation by Real-time PCR

HECTOR ANTONIO CRISTÓBAL

Microbial Resources

CABI UK Editorial

Año: 2015; p. 1 - 30

All living organisms regulate their activities through the activation or repression of expression of its genes. The genetic expression of a target gene is generally proportional to the number of copies of messenger RNA. Therefore, the detecting of specific cell products is a crucial fact when it comes to identify copies of mRNA when it is translated to form proteins. This measure allows obtaining data on the production of biological elements and the varying levels of expression of its genes in the cell in response to exposure from various effects. The identification of specific sequences (DNA or RNA) and detection of the genetic expression level represents important data in several areas such as in medicine, biotechnology, food industry, etc. Diverse molecular biology techniques are capable of detecting northern blotting, microarrays, capillary electrophoresis, real time PCR, etc. In order to obtain this information, real time PCR has become one of the most widely employed for gene quantification methods. This technique provides elevated data output with a high sensibility, detect sequence-specific, does not require post-amplification manipulation and can process a wide of samples numbers using modern equipment that avoids laboratory contamination. Nevertheless, several considerations like the choice of the strategy of quantification, fluorescent markers, as well as data interpretation, must be taken into account when working with real time PCR. This Chapter allows approaching the basic concepts on the real time PCR technique and the handling of obtained data; including  types of absolute and relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization, chemistries and applications in different field research?s. In addition, various steps for starting in real time PCR for determination the quantification of gene expression levels of 16SrDNA, gyrB, bgl-A, bgl and CspA from Shewanella sp. G5 cultures were evaluated and optimized.