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DNA Microarrays M. Ahmad Chaudhry, Ph. D. Director Microarray Facility University of Vermont Outline of the lecture • Overview of Micoarray Technology • Types of Microarrays • Manufacturing • Instrumentation and Softwares • Data analysis • Applications Microarray Development • Relatively young technology • Widely adopted • Mainly used in gene discovery Evolution & Industrialization • 1994- First cDNAs arrays were developed at Stanford University. • 1996- Commercialization of arrays • 1997-Genome-wide Expression Monitoring in S. cerevisiae What are Microarrays? • Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of genes (usually between 500-20,000) • Via a conventional DNA hybridization process, the level of expression/activity of genes is measured • Data are read using laser-activated fluorescence readers • The process is “ultra-high throughput” Why use Microarrays? • What genes are Present/Absent in a cell? • What genes are Present/Absent in the experiment vs. control? • Which genes have increased/decreased expression in experiment vs. control? • Which genes have biological significance? Why analyze so many genes? • Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function. • Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways The 6 steps of a DNA microarray experiment (1-3) 1. Manufacturing of the microarray 2. Experimental design and choice of reference: what to compare to what? 3. Target preparation (labeling) and hybridization The 6 steps of a microarray experiment (4-6) 4. Image acquisition (scanning) and quantification (signal intensity to numbers) 5. Database building, filtering and normalization 6. Statistical analysis and data mining GENE EXPRESSION ANALYSIS WITH MICROARRAYS DNA Chips Miniaturized, high density arrays of oligos (Affymetrix Inc.) Printed cDNA or Oligonucleotide Arrays Robotically spotted cDNAs or Oligonucleotides • Printed on Nylon, Plastic or Glass surface Affymetrix Microarrays Involves Fluorescently tagged cRNA • One chip per sample • One for control • One for each experiment Glass Slide Microarrays Involves two dyes/one chip • Red dye • Green dye • Control and experiment on same chip Gene Chip Technology Affymetrix Inc Miniaturized, high density arrays of oligos 1.28-cm by 1.28-cm (409,000 oligos) Manufacturing Process Solid-phase chemical synthesis and Photolithographic fabrication techniques employed in semiconductor industry Selection of Expression Probes Set of oligos to be synthesized is defined, based on its ability to hybridize to the target genes of interest 5’ 3’ Sequence Probes Perfect Match Mismatch Chip Computer algorithms are used to design photolithographic masks for use in manufacturing • • Each gene is represented on the probe array by multiple probe pairs Each probe pair consists of a perfect match and a mismatch oligonucleotide Photolithographic Synthesis Manufacturing Process Probe arrays are manufactured by light-directed chemical synthesis process which enables the synthesis of hundreds of thousands of discrete compounds in precise locations Lamp Mask Chip Affymetrix Wafer and Chip Format 20 - 50 µm 20 - 50 µm Millions of identical oligonucleotide probes per feature 49 - 400 chips/wafer 1.28cm up to ~ 400,000 features/chip Creating Targets mRNA Reverse Transcriptase cDNA in vitro transcription cRNA Target RNA-DNA Hybridization Targets RNA probe sets DNA (25 base oligonucleotides of known sequence) Non-Hybridized Targets are Washed Away Targets (fluorescently tagged) “probe sets” (oligo’s) Non-bound ones are washed away Target Preparation B Biotin-labeled transcripts B B B B Fragment (heat, Mg2+) B B B Fragmented cRNA IVT AAAA mRNA (Biotin-UTP Biotin-CTP) Wash & Stain Scan cDNA Hybridize (16 hours) ® GeneChip Expression Analysis Hybridization and Staining Array Hybridized Array cRNA Target Streptravidinphycoerythrin conjugate Instrumentation Affymetrix GeneChip System 3000-7G Scanner 450 Fluidic Station 640 Hybridization Oven Currently Available GeneChips B. subtilis Plasmodium/Anopheles Genome Array Barley Genome Array Porcine Genome Array Bovine Genome Array Rat Genome Arrays C. elegans Genome Array Rice Genome Array Canine Genome Array Soybean Genome Array Chicken Genome Array Sugar Cane Genome Array Drosophila Genome Arrays Vitis vinifera (Grape) Array E. coli Genome Arrays Wheat Genome Array Human Genome Arrays Xenopus laevis Genome Array Maize Genome Array Yeast Genome Arrays Mouse Genome Arrays Zebrafish Genome Array P. aeruginosa Genome Array Arabidopsis Genome Arrays Custom GeneChips Affymetrix offers over 120 prokaryotic arrays that are manufactured by Nimblegen Inc. Custom GeneChips are also available for both Eukaryotic and Prokaryotic systems. Quality Control Issues • RNA purity and integrity • cDNA synthesis efficiency • Efficient cRNA synthesis, labeling and fragmentation • Target evaluation with Test Chips GENE EXPRESSION ANALYSIS WITH MICROARRAYS DNA Chips Miniaturized, high density arrays of oligos (Affymetrix Inc.) Printed cDNA or Oligonucleotide Arrays Robotically spotted cDNAs or Oligonucleotides • Printed on Nylon, Plastic or Glass surface Microarray of thousands of genes on a glass slide steel Spotted arrays spotting pin chemically modified slides 384 well source plate 1 nanolitre spots 90-120 um diameter The process Building the chip: MASSIVE PCR PCR PURIFICATION and PREPARATION PREPARING SLIDES RNA preparation: CELL CULTURE AND HARVEST PRINTING Hybing the chip: POST PROCESSING ARRAY HYBRIDIZATION RNA ISOLATION DATA ANALYSIS cDNA PRODUCTION PROBE LABELING Building the chip Arrayed Library (96 or 384-well plates of bacterial glycerol stocks) Spot as microarray on glass slides PCR amplification Directly from colonies with SP6-T7 primers in 96-well plates Consolidate into 384-well plates Sample preparation Hybridization Binding of cDNA target samples to cDNA probes on the slide Hybridize for 5-12 hours Hybridization chamber 3XSSC HYB CHAMBER ARRAY LIFTERSLIP SLIDE LABEL SLIDE LABEL • Humidity • Temperature • Formamide (Lowers the Tm) Expression profiling with DNA microarrays cDNA “B” Cy3 labeled cDNA “A” Cy5 labeled Laser 1 Hybridization Laser 2 Scanning + Analysis Image Capture Image analysis • The raw data from a cDNA microarray experiment consist of pairs of image files, 16-bit TIFFs, one for each of the dyes. • Image analysis is required to extract measures of the red and green fluorescence intensities for each spot on the array. Image analysis GenePix Image analysis 1. Addressing. Estimate location of spot centers. 2. Segmentation. Classify pixels as foreground (signal) or background. 3. Information extraction. For each spot on the array and each dye • signal intensities; • background intensities; • quality measures. R and G for each spot on the array. Biological Question Data Analysis & Modelling Microarray Life Cycle Sample Preparation Microarray Detection Microarray Reaction Spotted cDNA microarrays Advantages • Lower price and flexibility • Simultaneous comparison of two related biological samples (tumor versus normal, treated versus untreated cells) • ESTs allow discovery of new genes Disadvantages • Needs sequence verification • Measures the relative level of expression between 2 samples Data Pre-processing Filtering – Background subtraction – Low intensity spots – Saturated spots – Low quality spots (ghost spots, dust spots etc) Normalization – Housekeeping genes/ control genes Affymetrix Software for Microarray Data Analysis • Microarray Suite 5 • Micro DB • Data Mining Tool (DMT) • NetAffx Affymetrix Microarray Suite - Data Analysis Absolute Analysis –whether transcripts are Present or not (uses data from one probe array experiment). Comparison Analysis –determine the relative change in transcripts (uses data from two probe array experiments). Intensities for each experiment are compared to a baseline/control. Microarray data analysis Scatter plots • Intensities of experimental samples versus normal samples • Quick look at the changes and overall quality of microarray Normal vs. Normal Normal vs. Tumor Lung Tumor: Up-Regulated Lung Tumor: Down-Regulated Microarray data analysis Supervised versus unsupervised analysis – Clustering: organization of genes that are similar to each other – Statistical analysis: how significant are the results? Hierarchical clustering • Unsupervised: no assumption on samples • The algorithm successively joins gene expression profiles to form a dendrogram based on their pair-wise similarities. Cluster analysis of genes in G1 and G2 Chaudhry et. al., 2002 Publicly Available Softwares GenMAPP Visualize gene expression data on maps representing biological pathways and groupings of genes. Microarray Applications • Identify new genes implicated in disease progression and treatment response (90% of our genes have yet to be ascribed a function) • Assess side-effects or drug reaction profiles • Extract prognostic information, e.g. classify tumors based on hundreds of parameters rather than 2 or 3. • Identify new drug targets and accelerate drug discovery and testing • ??? Microarray Technology - Applications • Gene Discovery– Assigning function to sequence – Discovery of disease genes and drug targets – Target validation • Genotyping – Patient stratification (pharmacogenomics) – Adverse drug effects (ADE) • Microbial ID The List Continues To Grow…. Microarray Future • Must go beyond describing differentially expressed genes • Inexpensive, high-throughput, genomewide scan is the end game for research applications • Protein microarrays (Proteomics) Microarray Future • Publications are now being focused on biology rather than technology • SNP analysis –Faster, cheaper, as accurate as sequencing –Disease association studies –Population surveys • Chemicogenomics –Dissection of pathways by compound application –Fundamental change to lead validation Microarray Future • Diagnostics – Tumor classification – Patient stratification – Intervention therapeutics Conclusion • Technology is evolving rapidly. • Blending of biology, automation, and informatics. • New applications are being pursued – Beyond gene discovery into screening, validation, clinical genotyping, etc. • Microarrays are becoming more broadly available and accepted. – Protein Arrays – Diagnostic Applications W.W.W resources • Complete guide to “microarraying” http://cmgm.stanford.edu/pbrown/mguide/ • http://www.microarrays.org – Parts and assembly instructions for printer and scanner; – Protocols for sample prep; – Software; – Forum, etc. • Animation: http://www.bio.davidson.edu/courses/genomics/chip/chip.html