Microarray

Transcription of DNA into RNA and the subsequent translation of messenger RNA into protein are the basic mechanisms by which cells mediate their growth, function and metabolism. After the human genome has been sequenced and annotated successfully a few years ago, the next step in functional genomics is to analyze the transcriptome, which can be defined as a complete collection of transcribed elements of the genome. In addition to messenger RNAs (mRNAs), the transcriptome can also represent non-coding regions of RNAs whose main functions are of structural and regulatory purposes. Alterations in the structure or expressions levels of any one of these RNAs or their proteins eventually will contribute to disease occurrences (Nelson et al., 2000). The use of microarray technologies to monitor gene expressions in organisms, cell lines, and human tissues has become very important in today’s biological research field (Schadt et al., 2000). The most well-known technologies developed to examine gene expressions of thousands of genes are the cDNA microarrays and oligonucleotide arrays. These two techniques are most famous for their ability to compare and contrast expression levels across various tissue types (Gibson et al., 2002). There are a few major differences between cDNA and oligonucleotide microarrays.

One difference is that cDNA microarrays only provide gene expression data in relative values as to that of absolute data values provided by oligonucleotide arrays. Another variation would be the difference in the design of the array since cDNA microarrays uses PCR-amplified cDNA fragments (ESTs) extracted from a sequenced cDNA library compared to oligonucleotide microarrays uses a series of 25-mer oligonucleotides to represent known or predicted open reading frames (Gibson et al., 2002; Lipshutz et al., 1999; Wilson et al., 2003).

2.4.1 cDNA Microarray Technology

cDNA microarrays is designed to monitor relative gene expression levels of thousands of genes in cells simultaneously. In a typical cDNA microarray chips, PCR-amplified cDNA fragments, also known as expression-sequence tags (ESTs), are spotted at high density, usually at 10-50 spots per mm², onto a glass microarray slide (Gibson et al., 2002). The two different mRNA samples derived independently will be transcribed into reverse-cDNA and labeled using two different fluorescents, which usually are a red fluorescent dye Cy5 and a green fluorescent dye Cy3. The labeled cDNA populations will then hybridized simultaneously to the glass microarray slide (Yang et al., 2002). Red and green laser beams will scan the microarray slides separately, and the signal intensity values observed from the two scans are calculated for individual cDNA spots by having the intensity levels of the experimental samples (Cy5) divided by the intensity levels of the reference sample (Cy3).

As a result, each derived gene expression level is a relative ratio for the cDNA spot in the sample (Figure 3).

The relative ratio obtained from cDNA microarrays has possessed a central idea that it is the change in relative level of expression that is of biological interesting. Genes with greater expression level do not mean that they have higher fluorescence intensities than genes with lower expression levels. The reason is that the fluorescence intensity is dependent on the length of the EST, the amount of label incorporated into the cDNA during the reverse transcription process, the preparation of DNA concentration for the particular clone and the efficiency of hybridization (GEO Website, 2005).

Figure 3: Overview Process of Making cDNA Microarray Chips (www.fao.org).

According to Claverie, the meaningful change in gene expression can be determined by the twofold induction or repression of experimental samples relative to the reference sample.

This rule, however, does not meet the standard statistical definitions of significance. As a result, genes in cDNA microarrays will be classified as “differentially expressed” only if they have shown at least a 2-fold change in expression (Claverie et al., 1999).

≥ 2

or

2

≤ 1

GeneB

GeneA

2.4.2 Oligonucleotide Microarray Technology

High-density oligonucleotide arrays are built, or synthesized in situ on a silicon chip by Affymetrix. Each gene is uniquely represented by 10 to 20 different nucleotides on a probe array. Probe synthesis takes place in a parallel fashion, in which an A, T, C, or G nucleotide will be added to multiple growing chains simultaneously. After having undergone through a series of photolithographic and combinatorial chemical process, each probe will reach its particular length of 25 nucleotides (Lipshutz et al., 1999; Schadt et al., 2000) (Figure 4).

Figure 4: Oligonucleotide microarray technology. (Lipshutz et al., 1999).

In order to prevent the possibility of having cross-hybridization with similar short sequences in transcripts rather than the one being probed, a partner probe is designed to be perfectly complementary to the target probe except that a single base in its centre will be purposely mutated, resulting in a mismatch probe (MM). As shown in Figure 5, each Mismatch (MM) probe, also known as partner probe, will be paired with a complementary Perfect Match (PM) probe, also known as reference probe, and these two probe pairs allow the quantization and subtraction of intensity signals caused by non-specific cross-hybridization (Gibson et al., 2002; Lipshutz et al., 1999; Schadt et al., 2000). In oligonucleotide arrays, the expression level of each gene is calculated based on the average of the differences between PM and MM, which means the derived value of each gene expression level is an absolute amount in oligonucleotide arrays rather than that of relative ratio in cDNA arrays (Schadt et al., 2000).

Figure 5: Oligonucleotide Probe Pair Design. Oligonucleotide probes are chosen based on composition design rules, whereas proves for eukaryotic organisms are chosen particularly from the 3’ end. The use of the PM–MM differences averaged across probe sets has reduced cross-hybridization problems and increased the quantitative accuracy (Lipshutz et al., 1999).