1. MICROARRAY

2. Microarray  A multiplex lab-on-a-chip  A 2D array on a solid substrate (Usually a glass slide or silicon thin-film cell) that assays large amounts of biological material using high- throughput screening methods  Types: DNA, RNA, Protein, Peptide, Tissue, Cellular, Antibody, Chemical compound, Carbohydrate  Focus on DNA microarray

3. DNA Microarray  To measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome  A simple concept: Dot Blot + Northern  Make probes for lots of genes (A massively parallel experiment) and put the probes on the filter  Make it tiny so you don’t need so much RNA from your experimental cells  Label the bulk RNA from samples of interest  Make quantitative measurements  Includes: cDNA microarrays, oligonucleotide microarrays and SNP microarrays

4. Types of DNA Microarray  Based on probe synthesis method 1. Spotted/cDNA 2. In situ synthesized/oligonucleotide/GeneChip  Based on color separation 1. Dual color/dual channel 2. Single color/single channel

5. cDNA microarray  Each DNA spot contains picomoles of a specific DNA sequence, known as probes/reporters/oligos  Probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs  The probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass  A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface  May be easily customized for each experiment  Laboratories can then generate their own labeled samples for hybridization, hybridize the samples to the array, and finally scan the arrays with their own equipment  Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target  Relatively low-cost microarray  May not be the same level of sensitivity compared to commercial oligonucleotide arrays

6. Custom Made Pin Spotter

7. Oligonucleotide Microarrays- GeneChip  Probes are short sequences designed to match parts of the sequence of known or predicted open reading frames  Oligonucleotide arrays are produced by printing short oligonucleotide sequences designed to represent a single gene or family of gene splice-variants by synthesizing this sequence directly onto the array surface instead of depositing intact sequences  Sequences may be longer (60-mer probes such as the Agilent design) or shorter (25-mer probes produced by Affymetrix) depending on the desired purpose  Affy uses photolithographic synthesis on a silica substrate where light and light-sensitive masking agents are used to "build" a sequence one nucleotide at a time across the entire array  Maskless Array Synthesis from NimbleGen Systems has combined flexibility with large numbers of probes

8. Microarray Spotting Techniques Printing can be done in one of three ways: (a) Photolithography, (b) Mechanical microspotting, (c) Ink jetting.

9. Two-Color Microarrays  Hybridized with cDNA prepared from two samples to be compared (e.g. diseased tissue versus healthy tissue) and that are labeled with two different fluorophores  Fluorescent dyes commonly: Cy3 (green, 570 nm) and Cy5 (670 nm, red)  The two Cy-labeled cDNA samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores after excitation with a laser beam of a defined wavelength. Relative intensities of each fluorophore may then be used in ratio-based analysis to identify up-regulated and down-regulated genes  Although absolute levels of gene expression may be determined in the two- color array in rare instances, the relative differences in expression among different spots within a sample and between samples is the preferred method of data analysis for the two-color system  Examples: Agilent with their Dual-Mode platform, Eppendorf with their DualChip platform for colorimetric Silverquant labeling, and TeleChem International with Arrayit

10. Single-Color Microarrays  Provide intensity data for each probe or probe set indicating a relative level of hybridization with the labeled target  Do not truly indicate abundance levels of a gene but rather relative abundance when compared to other samples or conditions  Data are more easily compared to arrays from different experiments so long as batch effects have been accounted for  Each RNA molecule encounters protocol and batch-specific bias during amplification, labeling, and hybridization phases of the experiment making comparisons between genes for the same microarray uninformative  Compared to the two-color system, twice as many microarrays are needed to compare samples within an experiment  Example: Affymetrix "Gene Chip", Illumina "Bead Chip", Agilent single-channel arrays, Applied Microarrays "CodeLink" arrays

11. Probe Design  Typically 25-70 bases  Shorter oligo probes: 1. More specific in hybridization 2. Better at discriminating perfect complementary sequences from sequences containing mismatches  Longer oligo probes: 1. More sensitive in binding cDNAs 2. Sometimes, multiple distinct oligonucleotide probes hybridizing different regions of the same transcript can be used to increase the signal-to-noise ratio

12. Optimal Oligonucleotide Probe  Specific enough to minimize cross-hybridization with nonspecific genes  Sensitive  Devoid of low-complexity regions (RepeatMasker & BLAST)  Should not form stable internal secondary structures (e.g. hairpin) [ DNA/RNA folding programs such as Mfold can help]  Oligo design should be close to the 3’ end of the gene because the cDNA collection is often biased to the 3’ end  All the probes should have an approximately equal melting temperature(Tm)  GC content of 45%-65%  Program: OligoWiz, OligoArray

13. Probe Set

14. Experiment Design  How many sample or replicate is necessary?  What should be the reference?

15. Experimental Replicates  In any experimental system, there is a certain amount of noise  2 identical processes yield slightly different results  In order to understand how much variation there is, it is necessary to repeat an experiment a number of independent times  Replicates allow us to use statistical tests to ascertain if the differences we see are real  Source of variation: Biological, Technical, Measurement error

16. Experimental Replicates  Technical replicates 1. Repeat a measurement with same RNA samples 2. Multiple probes for each gene: Within-slide, Between- slide 3. Increase precision  Biological replicates 1. Repeat a measurement with independent RNA samples 2. Provides better estimates of characteristic expression level 3. Increase accuracy

17. Two Color Experiment Design

18. Microarray Data Analysis Pipeline

19. Applications of DNA Microarray Microarray is currently used for many different purposes, including: Gene expression profiling via analyzing global mRNA levels Gene discovery Disease Diagnosis Classification of microbes and human microbial pathogens - GeneID Cellular responses to pathogens Drug and toxic exposures Drug discovery Tumor classification Single nucleotide polymorphism detection Detection of gene fusions Comparative genomic hybridization Alternative splicing detection (Exon junction array/Exon arrays/Tiling arrays) ChIP on Chip, DamID

20. References  Applied Mycology and Biotechnology- Volume 6, Bioinformatics- Chapter 1, Page 16