Microarray
Since the completion of the human genome project in 2001, many different technologies have evolved to better aid in the analysis of the sequenced genes. Among those is the microarray technology which enables researches to analyse the expression of many genes in a single reaction quickly and efficiently. Now the regions of DNA associated with the fundamentals of protein production and the causes of genetic anomalies can be studied (Govindarajan, Duraiyan, Kaliyappan, & Palanisamy, 2012; Jaluria, Konstantopoulos, Betenbaugh, & Shiloach, 2007; Miller & Tang, 2009; Russo, Zegar, & Giordano, 2003).
Principle and Mechanism
The concept of the microarray was derived from Southern blotting and is based on conducting parallel expression of multiple genes in one reaction. A basic microarray experiment is composed of a chip/microscope glass/nylon membrane containing many wells with different DNA sequences hybridized on it.
The mechanism involves the hybridization of an mRNA molecule to its complementary source DNA. Many DNA samples are used to construct an array called probes that are immobilized onto the plate containing either 34 or 96 wells; each well has a size of less than 200 microns. These probes are known DNA sequences. The probes may also be cDNA or oligonucleotides depending on the unknown samples. Quantification of the mRNA strands bound to their complementary DNA or cDNA probes indicates the expression level of that mRNA. The data can then be assimilated for the function of the mRNA based on it hybridizing with the DNA probe as well as which tissue expresses high levels of this particular mRNA to deduce its function within the tissue. Generally, the sample mRNA or sequence to be tested is labelled with a probe. The exact steps can be summarized as first extracting mRNA, then conducting a reverse-transcription PCR to cDNA, labelling the cDNA, hybridizing the cDNA to the probes on the plate, and scanning the plates to determine and quantify the fluorescence emission. Most common labelling of cDNA is using fluorochrome dyes Cy3 (green) and Cy5 (red). An integral step within the process is normalization of the intensity of the fluorescence after obtaining the results (Govindarajan et al., 2012; Jaluria et al., 2007; Miller & Tang, 2009; Russo et al., 2003).
Figure 1. This figure shows the expected results after conducting the microarray experiment. Notice the different colours which represent the reference and test samples hybridizing to their complementary strands and the intensity of the colours representing the expression levels (Miller & Tang, 2009).
Types
The types of microarrays can be categorized based on the type of immobilized probe into:
1. DNA microarray
2. Protein microarray
3. Peptide microarray
4. Cellular microarray
5. Tissue microarray
6. Phenotype microarray
7. Antibody microarray
8. Reverse phase protein microarray
The types may also be classified according to the objective of the experiment into:
1. Expression analysis: usually used to compare expression of diseased vs. normal genes.
2. Mutation analysis: usually used to identify point mutations or SNPs.
3. Comparative genomic analysis: usually used to measure the expression level is segments of chromosomes related to diseases.
Finally, the categorization may also be based on the chip/channel itself and the experimental design into:
1. Single channel approach
2. Multiple channel approach
3. Speciality approaches such as using beads (Govindarajan et al., 2012; Jaluria et al., 2007; Miller & Tang, 2009; Russo et al., 2003).
Figure 2. Representation of single channel (one colour) microarray vs. multiple channels (two colours) microarray emphasizing that the overall steps are the same; however, there are differences in the method of detection whether it is labelling the reference and test samples differently (multiple channels) or applying the same label (single channel) (Miller & Tang, 2009).
Advantages
1. Provides data for large numbers of genes
2. Can be conducted in one reaction instead of multiple reactions
3. Results obtained are fast and reaction is relatively easy
4. Can easily provide links between disease progression and associated genes and their expression levels
5. Not restricted to DNA samples to study expression levels (Govindarajan et al., 2012; Jaluria et al., 2007; Miller & Tang, 2009; Russo et al., 2003).
Disadvantages
1. Chips/plates are expensive to produce
2. The plates have a short shelf life
3. Analysis of the results is complicated and a lengthy procedure (Govindarajan et al., 2012; Jaluria et al., 2007; Miller & Tang, 2009; Russo et al., 2003).
Applications
Microarray technology has a wide range of applications within every industry, but the most promising applications are:
Gene discovery: through microarrays, gene function and expression levels may be determined according to the variable cellular conditions.
Drug discovery: microarrays are extensively used in the field of pharmacogenomics which is the study of individualized therapy based on treatments dependant on the genetic profile of an individual. Through identification of diseased genes, the proteins synthesized by them can be isolated and targeted by drugs.
Disease diagnosis: genes associated with the progression of diseases may be assessed for in one reaction to determine which are pivotal. For example, cancer has been classified based on its genetic pattern instead of just based on the tumor growth.
Toxicological research: the changes in the genetic profiles of an individual based on exposure to toxins can be reported through the study of toxicogenomics which is dependent on microarray technology (Govindarajan et al., 2012; Jaluria et al., 2007; Miller & Tang, 2009; Russo et al., 2003).
