Applications of Molecular Genetics to the Study of Asthma.

Asthma is a multifactorial disease. This fact, associated to the diversity of asthma phenotypes, has made difficult to obtain a clear pattern of inheritance. With the huge development of molecular genetics technologies, candidate gene studies are giving way to different types of studies from the genomic point of view.These approaches are allowing the identification of several genes associated with asthma. However, in these studies, there are some conflicting results between different populations and there is still a lack of knowledge about the actual influence of the gene variants. Some confounding factors are, among others, the inappropriate sample size, population stratification, differences in the classification of the phenotypes, or inadequate coverage of the genes.To confirm the real effect of the reported associations, it is necessary to consider both the genetic and environmental factors and perform functional studies that explain the molecular mechanisms mediating between the emergence of gene variants and the development of the disease.The development of experimental techniques opens a new horizon that allows the identification of major genetic factors of susceptibility to asthma. The resulting classification of the population groups based on their genetic characteristics, will allow the application of specific and highly efficient treatments.

Introduction to the Gene Expression Analysis.

In 1941, Beadle and Tatum published experiments that would explain the basis of the central dogma of molecular biology, whereby the DNA through an intermediate molecule, called RNA, results proteins that perform the functions in cells. Currently, biomedical research attempts to explain the mechanisms by which develops a particular disease, for this reason, gene expression studies have proven to be a great resource. Strictly, the term "gene expression" comprises from the gene activation until the mature protein is located in its corresponding compartment to perform its function and contribute to the expression of the phenotype of cell.The expression studies are directed to detect and quantify messenger RNA (mRNA) levels of a specific gene. The development of the RNA-based gene expression studies began with the Northern Blot by Alwine et al. in 1977. In 1969, Gall and Pardue and John et al. independently developed the in situ hybridization, but this technique was not employed to detect mRNA until 1986 by Coghlan. Today, many of the techniques for quantification of RNA are deprecated because other new techniques provide more information. Currently the most widely used techniques are qPCR, expression microarrays, and RNAseq for the transcriptome analysis. In this chapter, these techniques will be reviewed.

Validation of Reference Genes in mRNA Expression Analysis Applied to the Study of Asthma.

The quantitative Polymerase Chain Reaction is the most used technique for the study of gene expression. To correct putative experimental errors of this technique is necessary normalizing the expression results of the gene of interest with the obtained for reference genes. Here, we describe an example of the process to select reference genes. In this particular case, we select reference genes for expression studies in the peripheral blood mononuclear cells of asthmatic patients.

Interactions of DNA and Proteins: Electrophoretic Mobility Shift Assay in Asthma.

Electrophoretic mobility shift assays (EMSA) are used to characterize interactions between nucleic acids and proteins in native conditions. This is based on the fact that the electrophoretic mobility of a nucleic acid becomes slower when it forms complexes with proteins. There are many different variants and applications of this methodology. In this chapter we describe a detailed EMSA protocol applied to the study of asthma.