**2. Microarrays and drug discovery**

### **2.1 Microarray technology: role, types, and applications**

The microarray technology is powerful though early on it has had some limitations due to its high costs. However, in recent years, it has become more affordable with the availability of commercial microarray chips and platforms. Thus, this technology has moved from research laboratories to clinical applications. In recent years, microarrays have played significant roles in drug discovery. A large number of studies have demonstrated that microarray datasets not only allow for rapid and direct analysis of large amounts of biological information, but these also promote identification of potential biomarkers for various diseases [24–28]. Furthermore, microarray datasets can potentially determine the appropriate drug dose that can maximize its therapeutic effect. In clinical trials, microarrays can be used for early detection of any toxicity or any side-effects of a drug or a drug dose in order to provide rapid, sensitive, and safe treatments. Moreover, microarrays play important roles in pharmacogenomics by allowing for identification of associations between responses to drug treatment and a patient's genetic profile [28, 29], as well as for selecting the most appropriate new candidate drugs for clinical trials.

There are several types of microarrays, including DNA microarrays, microRNA arrays, chemical compound microarrays, antibody microarrays, protein microarrays, tissue microarrays, and carbohydrate arrays. In clinical research, DNA microarrays are often used for novel biomarker discovery [30]. Among other applications of DNA/RNA microarrays are the following: 1) identification of differential gene expression, 2) analysis of mutations, 3) screening of single nucleotide polymorphisms (SNPs), 4) determination of methylation, acetylation, and alternative splicing, and 5) comparative genomic hybridization [31–34].

Microarrays consist of hundreds to thousands of DNA, RNA, oligonucleotides, or other probe molecules that are immobilized in an array format onto a solid support surface, such as microscope glass slides, silicon chips, or nylon membranes, and then exposed to labeled samples carrying corresponding target molecules to allow for simultaneous detection of nucleic acid/protein/antibody/other targets. Typically, a single probe is at one-time leading to a microarray with hundreds of thousands of different oligonucleotide sequences complementary to distinct fragments of known DNA or RNA sequences [35]. Components of a DNA or an RNA sample loaded onto a slide/chip/membrane will hybridize specifically to their complementary probes, and the fluorescence intensity will correspond to the amount of DNA or RNA of a given gene in a sample [36].

Microarrays are processed in either "one-color" or "two-color" formats. In a one-color format, a single RNA sample is labeled with a fluorophore, such as cyanine-3 (Cy-3) or cyanine-5 (Cy-5) prior to hybridization, and the intensity of the fluorophore is determined [37]. Whereas a two-color microarray capitalizes on a competitive hybridization (**Figure 1**). In this format, a single nucleic acid sample is labeled with a green dye, while a related sample is labeled with a red dye. Following hybridization and removal of unbound nucleic acids, a laser scanner will detect those red- and green-labeled molecules. The intensity of each colored spot on an array is determined, and the red/green ratio is determined [38].

**57**

**Table 1.**

(CGH)

*Microarrays and NGS for Drug Discovery DOI: http://dx.doi.org/10.5772/intechopen.96657*

including microarrays, are listed in **Table 1**.

**2.2 Droplet Digital PCR (ddPCR) and microarrays**

ChIP microarrays Drug development

Splice variants Pharmacogenomics

Genotyping Drug discovery

Comparative genomic hybridization

*Available techniques for drug discovery.*

Several microarray technologies have been developed using various platforms that have been optimized to maximize reproducibility and accuracy of findings [39]. For example, Affymetrix GeneChip microarrays are manufactured using photolithography that utilize oligonucleotide probes. This system has the capability of monitoring expression of every gene in a genome. In fact, Affymetrix GeneChips have been used for genotyping, copy number analysis, transcriptome analysis, and miRNA profiling. On the other hand, Agilent oligonucleotide microarrays are based on inkjet technology for *in situ* manufacturing of probes, wherein actual probe sequences are used as linkers in order to extend these probes to provide higher specificity [39]. Whereas, Illumina BeadArrays are based on patterned substrates for high-density detection of target nucleic acids using silica microbeads [40]. Some of the common available techniques used in drug development efforts,

The Droplet Digital PCR (ddPCR) is a recent technology that is commercially available, capitalizing on the use of *Taq* polymerase in a standard PCR reaction in order to amplify a target DNA fragment from a complex sample using pre-validated primers or primer/probe assays [61, 62]. Galbiati et al. have proposed a workflow that combined a microarray assay with ddPCR for both detection and quantification of circulating tumor DNA mutations in colon cancer patients [63]. This approach is useful for the development of reliable non-invasive biomarkers for RAS and BRAF mutations, identifying a target mutation, and providing clinically relevant information. Microarray analysis and ddPCR data have identified mutations in primary breast tumors from female patients treated with adjuvant mono-tamoxifen therapy [64]. Moreover, using microarray and ddPCR, it is observed that epidermal growth factor receptor (EGFR) expression can be used as a prognostic biomarker in patients with oropharyngeal squamous cell carcinoma, as it is associated with smoking status [65]. In another study, microarray analysis of uterine tissue, along with validation using ddPCR has allowed for observing downregulation of genes in pathways of

**Techniques Applications References**

Pharmacogenomics Gene discovery Gene expression profiling

Drug discovery Biomarker identification Polymorphism/SNP detection Drug target identification

Pharmacogenomics Environmental monitoring

Vaccine candidate identification

Vaccine candidate identification

Drug resistance

Gene discovery Biomarker identification Clinical application

[41–46]

[47–52]

[53–57]

[29, 52, 58–60]

*A general workflow for a typical two-color microarray experiment.*

*Microarrays and NGS for Drug Discovery DOI: http://dx.doi.org/10.5772/intechopen.96657*

*Drug Design - Novel Advances in the Omics Field and Applications*

splicing, and 5) comparative genomic hybridization [31–34].

amount of DNA or RNA of a given gene in a sample [36].

array is determined, and the red/green ratio is determined [38].

of DNA/RNA microarrays are the following: 1) identification of differential gene expression, 2) analysis of mutations, 3) screening of single nucleotide polymorphisms (SNPs), 4) determination of methylation, acetylation, and alternative

or other probe molecules that are immobilized in an array format onto a solid support surface, such as microscope glass slides, silicon chips, or nylon membranes, and then exposed to labeled samples carrying corresponding target molecules to allow for simultaneous detection of nucleic acid/protein/antibody/other targets. Typically, a single probe is at one-time leading to a microarray with hundreds of thousands of different oligonucleotide sequences complementary to distinct fragments of known DNA or RNA sequences [35]. Components of a DNA or an RNA sample loaded onto a slide/chip/membrane will hybridize specifically to their complementary probes, and the fluorescence intensity will correspond to the

Microarrays are processed in either "one-color" or "two-color" formats. In a one-color format, a single RNA sample is labeled with a fluorophore, such as cyanine-3 (Cy-3) or cyanine-5 (Cy-5) prior to hybridization, and the intensity of the fluorophore is determined [37]. Whereas a two-color microarray capitalizes on a competitive hybridization (**Figure 1**). In this format, a single nucleic acid sample is labeled with a green dye, while a related sample is labeled with a red dye. Following hybridization and removal of unbound nucleic acids, a laser scanner will detect those red- and green-labeled molecules. The intensity of each colored spot on an

Microarrays consist of hundreds to thousands of DNA, RNA, oligonucleotides,

**56**

**Figure 1.**

*A general workflow for a typical two-color microarray experiment.*

Several microarray technologies have been developed using various platforms that have been optimized to maximize reproducibility and accuracy of findings [39]. For example, Affymetrix GeneChip microarrays are manufactured using photolithography that utilize oligonucleotide probes. This system has the capability of monitoring expression of every gene in a genome. In fact, Affymetrix GeneChips have been used for genotyping, copy number analysis, transcriptome analysis, and miRNA profiling. On the other hand, Agilent oligonucleotide microarrays are based on inkjet technology for *in situ* manufacturing of probes, wherein actual probe sequences are used as linkers in order to extend these probes to provide higher specificity [39]. Whereas, Illumina BeadArrays are based on patterned substrates for high-density detection of target nucleic acids using silica microbeads [40].

Some of the common available techniques used in drug development efforts, including microarrays, are listed in **Table 1**.
