Gene Expression Analysis : Determining messenger RNA levels

The tools of biotechnology not only allow the study of gene structure, but also provide ways of analyzing the mRNA and protein products of gene expression.
Determining messenger RNA levels
mRNA levels are usually determined by the hybridization of labeled probes to either mRNA itself or to cDNA produced from mRNA. [Note: Amplification by PCR of cDNA made from mRNA by retroviral reverse transcriptase (RT) is referred to as RT-PCR.]
1. Northern blots
Northern blots are similar to Southern blots , except that the sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized with a radiolabeled probe. The bands obtained by autoradiography give a measure of the amount and size of the mRNA molecules in the sample.
2. Microarrays: DNA microarrays contain thousands of immobilized ssDNA sequences organized in an area no larger than a microscope slide.
These microarrays are used to analyze a sample for the presence of gene variations or mutations (genotyping) or to determine the patterns of mRNA production (gene expression analysis), analyzing thousands of genes at the same time. For genotyping analysis, the sample is from genomic DNA. For expression analysis, the population of mRNA molecules from a particular cell type is converted to cDNA and labeled with a fluorescent tag (Fig. 1). This mixture is then exposed to a gene (or, DNA) chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene.

The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the sample. DNA microarrays are used to determine the differing patterns of gene expression in two different types of cell (for example, normal and cancer cells; see Fig. 1). They can also be used to subclassify cancers, such as breast cancer, to optimize treatment. [Note: Microarrays involving proteins and the antibodies or other proteins that recognize them are being used to identify biomarkers to aid in the diagnosis, prognosis, and treatment of disease based on a patient’s protein expression profile. Protein (and DNA) microarrays are important tools in the development of personalized (precision) medicine in which the treatment and/or prevention strategies consider the genetic, environmental, and lifestyle variations among individuals.]

Figure 1: Microarray analysis of gene expression using DNA (gene) chips. [Note: Protein chips are also used.] mRNA = messenger RNA; cDNA = complementary DNA.