Measuring specific gene expression patterns
While targeted arrays are now available, such arrays still probe between 50 and 500 genes depending on the array (e.g., Kim et al., 2015). When using gene expression to assess cell function, particularly phenotype, many studies tend to focus on a subset of genes that are known to be indicative of the differentiated function of that particular cell type. It typically yields important information and can significantly reduce associated costs, as the gene expression profile is typically assessed using three to five genes (e.g., Miron et al., 2010).
Measuring individual genes is based on reverse transcription polymerase chain reaction (RT-PCR) methodology. Conventional RT-PCR involves creation of a cDNA library from mRNA using reverse transcription, followed by PCR amplification, and electrophoresis of the products on gels (Roth, 2002). The bands of the amplified products are visualized with DNA-binding dyes such as ethidium bromide. By measuring the intensity of the endpoint products on the gel, this technique is only semiquantitative and is not precise enough to compare the gene expression between multiple treatment groups. The main limitation of RT-PCR is that the most accurate phase for measuring differences in mRNA copy is during the linear phase, which conventional RT-PCR does not measure (Roth, 2002).
This limitation leads to the development of another PCR-based approach that specifically measures amplification of the target during the linear phase (Wong and Medrano, 2005) (Fig. 6.1). This approach is referred to as reverse transcriptase
Figure 6.1 Typical RT-qPCR amplification curves. The most accurate phase for measuring a PCR product is during the linear phase of the curve, when the conditions are ideal and should result in the product doubling every cycle.
real-time PCR, or reverse transcriptase quantitative PCR. The commonly accepted abbreviation of real-time PCR is RT-qPCR, which we will use for the remainder of the chapter. Detection of amplified targets in real time is possible through the addition of a fluorescent reporter molecule in the reactions, with increased fluorescence corresponding with increased cDNA product (Heid et al., 1996). As RT-qPCR measures the amount of product in the linear phase of amplification, it becomes possible to measure the initial quantity of the template in the sample based on the knowledge that the template will double approximately every cycle.
While PCR-based methods are specific and highly appropriate for biomaterial research, they can be considered less encompassing compared to the microarray technology. With RT-PCR or RT-qPCR, the user is selecting which genes will be analyzed, and as such, important genes that maybe up- or downregulated may not be studied. However, it is important to understand that differentiation markers are well defined in many cell and tissue systems, and PCR-based methods are still a powerful method for analyzing changes in cell phenotype.
Localizing the expression of genes of interests
Whereas DNA microarrays and RT-qPCR are methods that measure the amount of specific mRNA levels in samples, neither allows the mRNA signal to be localized within tissues. Using fluorescent, chemi-luminescent, or radioactive-labelled nucleic acid probes, in situ hybridization allows the localisation of specific mRNA sequences on tissue cross-sections.
In situ hybridization can be seen as complementary to immunohistochemistry; both can reveal the distribution of specific mRNA (in situ hybridization) or protein (immu- nohistochemistry) within a tissue, although neither technique can quantify the amount of the targeted mRNA or protein present in the tissue. As it is a powerful technique to assess the distribution of specific mRNAs to specific cell populations, in situ hybridization is often used for in vivo experiments.