Microarray: An Introduction

Gene Background Gene: a region of the human genome coding for a protein Biologists interested in gene function and disease Studying multiple genes is time consuming and expensive

• Different cell types (e.g. muscle cells, fibroblasts) § Genes are differentially expressed in... • Different cell types (e.g. muscle cells, fibroblasts) • Environmental conditions (e.g. heat shock, nutrient deprivation) • Developmental phases • Cell-cycle stages (e.g. G1 phase) • Disease states (e.g. tumor cells, virus-infected cells)

Gene expression is primarily regulated at the level of transcription § Hence, the number of mRNA copies in a cell for a particular gene is a good indicator of that gene’s expression (number of proteins) § Dynamic range of mRNA levels: • Highly expressed genes can have up to 9400 mRNA copies per cell • Poorly expressed genes can have < 0.3 mRNA copies per cell

Regulation of Gene Expression m-RNA stability Control Translation control RNA process control NUCLEAS Cytosol Primary RNA transcript m-RNA m-RNA Protein DNA Active protein Transcriptional Control Transport Control Post-translational Control

Traditional Methods Northern Blotting Single RNA isolated Probed with labeled cDNA Reverse Transcription – Polymerase Chain Reaction (RT-PCR) Real time or quantitative RT-PCR Each technique measures the m-RNA levels of one or a few genes at a time

Microarray Technology New Technology (first paper: 1995) Allows study of thousands of genes at same time Tools used to measure simultaneously the transcription level of every gene in a cell

Advantages of DNA microarray experiments Lack information a priori of which genes may have altered mRNA levels in a particular experimental condition Knowledge of patterns of genes with altered mRNA levels is usually more informative than knowledge of single genes with altered mRNA levels Use statistical analysis of microarray data to test hypotheses or generate new hypotheses

Microarray Technology Quantitative Measurement of Gene Expression Also known as DNA microarrays, DNA arrays, DNA chips, gene chips, … Whatever the name, their use is effectively transforming a living from a black box into a transparent box.

Applications of Microarray Technology

Applications of Microarray Technology What type of data analysis is required to: Identify Genes expressed in different cell types (e.g. Liver vs finger) Learn how expression levels change in different developmental stages (embryo vs. adult) Learn how expression levels change in different developmental stages (cancerous vs non-cancerous) Learn how groups of genes inter-relate (gene-gene interactions) Identify cellular processes that genes participate in (structure, repair, metabolism, replication, … etc) Applications covered only as example contexts, emphasis is on analysis methods

Microarray Technology Types of microarray technologies and how they work Outputs of microarrays Image Analysis required to transform output to gene expression matrices

Fabrications of Microarrays Size of a microscope slide Images: http://www.affymetrix.com/

The Mechanics of a DNA Microarray Experiment Isolate mRNA from cell cultures Reverse Transcribe mRNA into cDNA Label cDNA or cRNA by incorporating fluorescently-labeled nucleotides Hybridize labeled cDNA (or cRNA) to DNA microarray Wash and scan microarray in confocal laser scanner Analyze data

Differing Conditions Ultimate Goal: Helps to: Understand expression level of genes under different conditions Helps to: Determine genes involved in a disease Pathways to a disease Used as a screening tool

Gene Conditions Cell types (brain vs. liver) Developmental (fetal vs. adult) Response to stimulus Gene activity (wild vs. mutant) Disease states (healthy vs. diseased)

Expressed Genes Genes under a given condition mRNA extracted from cells mRNA labeled Labeled mRNA is mRNA present in a given condition Labeled mRNA will hybridize (base pair) with corresponding sequence on slide

Constructing DNA Microarrays A DNA microarray is a collection of DNA probes separated in a regular array atop a solid support (glass slide, silicon chip, etc) Affymetrix oligonucleotide microarrays Short (25mers) oligonucleotide probes synthesized on the array cDNA microarrays cDNA PCR products printed on coated glass slides Oligonucleotide microarrays Long (70 - 100mers) oligonucleotide probes are printed on coated glass slides

Custom Arrays Mostly cDNA arrays 2-dye (2-channel) RNA from two sources (cDNA created) Source 1: labeled with red dye (Cy5) Source 2: labeled with green dye (Cy3)

Two Channel Microarrays Microarrays measure gene expression Two different samples: Control (green label) Sample (red label) Both are washed over the microarray Hybridization occurs Each spot is one of 4 colors

Building the chip Ngai Lab arrayer , UC Berkeley Print-tip head

well plate Glass Slide cDNA clones Pins collect cDNA from wells Contains cDNA probes cDNA clones Print-tip group 1 Glass Slide Array of bound cDNA probes In this case: 4x4 blocks = 16 print-tip groups Print-tip group 7

Microarray Image Analysis Microarrays detect gene interactions: 4 colors: Green: high control Red: High sample Yellow: Equal Black: None Problem is to quantify image signals

A two-channel microarray experiment

Microarray Animations Davidson University: http://www.bio.davidson.edu/courses/genomics/chip/chip.html Imagecyte: http://www.imagecyte.com/array2.html