All living organisms are composed of cells. As a functional unit, each cell can make copies of itself, and this process depends on a proper replication of the genetic material known as deoxyribonucleic acid (DNA). DNA contains genes, and each structural gene functions by transcribing it into the corresponding messenger RNA (mRNA) using DNA as a template and ultimately translating into the corresponding protein using mRNA as a template . The abundance and stability of proteins determine the functions of a cell. Thus, the function or activity of a gene is reflected by synthesis of mRNA (transcription) or protein (translation). DNA micro array technology measures the activity of genes at a transcriptional level.
DNA microarrays (sometimes called DNA chips) are in general characterized by a structured immobilization of DNA targets in the free nucleic acid samples on planar solid supports, on which different types of nucleic acids with known sequences (known as “probes”) are fixed. A probe may be derived from complementary DNA (cDNA), polymerase chain reaction (PCR) products, or synthetic oligomers. In general, applications of DNA microarray technology broadly include :
(1) gene expression analysis (transcription analysis), which analyzes the transcriptional activity of genes through hybridization between DNA targets and probes;
(2) genotyping with oligonucleotide arrays, which is based on the notion of combining the complete sequence of a DNA sample by presenting all possible sequences as a complement on the chip (Drmanac et al., 2002);
(3) measurement of enzyme activities on immobilized DNA, which is based on the finding that DNA-modifying enzymes are capable of acting on immobilized DNA templates or oligonucleotides (Bier et al., 1996a; Bier et al., 1996b; Buckle et al., 1996);
(4) PCR on the chip, which was first described in 2000 (Adessi et al., 2000); and (5) transcription on chip, which shows the transcription of a complete gene into mRNA on the chip (Steffen et al., 2005).
The procedure of a DNA microarray experiment includes multiple steps from sample preparation to data analysis, among which hybridization is a central process. The sample that contains the targets to
be investigated is added to the DNA chip to allow their binding to the probes fixed on the chip, resulting in a characteristic-binding pattern representing the levels of gene expression of the sample. The sample itself is labeled prior to the hybridization, and the most often used labels are fluorescence labels that allow detection of the binding event. After the hybridization, the chip is washed and then fluorescence intensities on the chip are read and recorded by a scanning or imaging device. Raw data
reflecting the fluorescence intensities are statistically analyzed and often shown by fold changes as compared to control.
In our laboratory, we performed DNA microarray experiments to study gene expression profiling in mouse leukemia cells. Here is an example of the procedures for carrying out the microarray periments. Briefly, cells are dissolved in RNAlater (Ambion, Austin, TX, USA) and homogenized in RLT Buffer (RNeasy Micro Kit; Qiagen, Valencia, CA, USA). Total RNA is isolated by following the protocol for the RNeasy Micro Kit, and quality is assessed using a 2100 Bioanalyzer instrument and RNA 6000 PicoLabChip assay (Agilent Technologies, Palo Alto, CA, USA). Utilizing the GeneChip Whole Transcript Sense Target Labeling Assay kit (Affymetrix, Santa Clara, CA, USA), 100–300 ng of total RNA undergoes reverse transcription with random hexamers tagged with T7 sequence. The double
stranded cDNA that is generated is then amplified by T7 RNA polymerase to produce cRNA. Second cycle first strand cDNA synthesis then takes place, incorporating dUTP, which is later used as sites where fragmentation occurs by utilizing a uracil DNA glycosylase and apurinic/apyrimidinic
endonuclease 1 enzyme mix. The fragmented cDNA is then labeled by terminal transferase, attaching a biotin molecule using Affymetrix proprietary DNA Labeling Reagent. Approximately 2.0 µg of fragmented and biotin labeled cDNA is then hybridized onto a Mouse Gene ST 1.0 Array (Affymetrix, Santa Clara, CA, USA) for 16 hours at 45°C. Posthybridization staining and washing are performed according to the manufacturer’s protocols using the Fluidics Station 450 instrument (Affymetrix). Finally, the arrays are scanned with a GeneChip Scanner 3000. Images are acquired and CEL files generated, which are then used .
Advantages and Disadvantages
One of the biggest advantages of DNA microarray technology is that it can evaluate simultaneously the relative expression of thousands of genes by using small amounts of materials, providing gene signatures for particular disease situations. In addition, the procedures can easily be automated.
Furthermore, the capacity of measurement of gene expression by DNA microarray is huge, allowing researchers to take the expression of all genes from an individual into consideration for disease analysis in so- called “personalized medicine”.
One of the major disadvantages of DNA microarray technology is that it only evaluates gene expression at a transcriptional, but not translational, level, as posttranscriptional modifications (such as phosphorylation) often play significant roles in the regulation of protein functions. In addition, DNA microarray technology is still not mature enough for decision making based on the microarray data. In this book, we introduce our new way of interpreting and analyzing microarray data, which will hopefully bring us closer to success in decision making using the information obtained through DNA microarray technology.