1. Microarray: An Introduction

2. 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

3. • 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)

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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.

10. Applications of Microarray Technology

11. 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

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

13. Fabrications of Microarrays
Size of a microscope slide
Images:

15. 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

16. 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

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

18. 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

19. 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 ( mers) oligonucleotide probes are printed on coated glass slides

20. 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)

21. 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

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

23. 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

25. 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

26. A two-channel microarray experiment

28. Microarray Animations
Davidson University:
Imagecyte: