Introduction to microarray technology Lecture 17, Statistics 246 March 18, 2004 1
Outline A little background Types of microarrays cdna arrays Affymetrix GeneChips 2
Uses and types of microarrays Microarrays are currently used to do many different things: to detect and measure gene expression at the mrna or protein level; to find mutations and to genotype; to (re)sequence DNA; to locate chromosomal changes (CGH = comparative genomic hybridization), and more. There are many different ways to do these things without microarrays, but microarrays promise a highthroughput approach to the tasks. There are many different types of microarrays (called platforms) in use, but all have a high density and number of biomolecules fixed onto a well-defined surface. Low density means 100s (e.g. protein antibodies), medium density would be 1000s to 10s of 1000s (e.g. cdna arrays), and high-density is 100s to 1000s of 1000s, i.e.millions (e.g. short oligonucleotide arrays). In general there are five basic aspects of microarrays: a) coupling biomolecules to a platform; b) preparing samples for detection; c) hybridization; d) scanning; and e) analyzing the data. Obviously we re interested in e), but without some knowledge of a) to d), we d be dangerous. 3
Nucleic acid hybridization: here DNA-RNA 4
The rudiments of hybridization kinetics can be helpful 5
Rudiments, completed 6
A knolwedge of the Polymerase Chain Reaction (PCR) can be helpful This reaction is used to amplify specific DNA sequences in a complex mixture when the ends of the sequence are known. The source is heat-denatured into single strands. Two synthetic oligonucleotides complementary to the 3 ends of the segment of interest are added in great excess to the denatured DNA and the temperature is lowered to 50-60 C or even lower. The genomic DNA remains denatured, because the complementary strands are at too low a concentration to encounter each other during the period of incubation, but the specific oligonucleotides, which are at a very high concentration, hybridize with their complementary sequences in the genomic DNA. 7
PCR, ctd The hybridized oligos then serve as primers for DNA chain synthesis, which begins upon addition of a supply of dntps and a temperature resistant polymerase such as that from Thermus aquilus (a bacterium that lives in hot springs). This enzyme, called Taq polymerase, can extend primers at temperatures up to 72 C. When synthesis is complete, the whole mixture is heated further (to 95 C) to melt the newly formed duplexes. When the temperature is lowered again, a new round of synthesis takes place because excess primer is still present. Repeated cycles of synthesis (cooling) and melting (heating) quickly amplify. 8
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The cdna and short (25 bp) oligo technologies in brief. Long (60-75 bp) oligo arrays are more like the cdna ones 10
cdna clones (probes) cdna arrays in summary laser 2 excitation laser 1 scanning printing PCR product amplification purification mrna target) emission overlay images and normalise 0.1nl/spot microarray Hybridise target to microarray analysis 11
Affymetrix GeneChips in summary (details slightly out of date) * * * * * 12
cdna microarrays on glass slides A little more detail An overview of the Brown-De Risi- Iyer technology, based on the 2000 CSH Microarray Course notes, Nature Genetics Supp, Jan 1999, two books edited by M Schena: DNA Microarrays, A Practical Approach, OUP 1999, and Microarray Biochip Technology, Eaton Publishing, 2000, DNA Arrays or Analysis of Gene Expression by M. Eisen and P. Brown, and the experiences of my colleagues. 13
cdna arrays: history cdna microarrays have evolved from Southern blots, with clone libraries gridded out on nylon membrane filters being an important and still widely used intermediate. Things took off with the introduction of non-porous solid supports, such as glass - these permitted miniaturization - and fluorescence based detection. Currently, up to about 30,000 cdnas are spotted onto a microscope slide. 14
Building the Chip: cdna arrays: the process MASSIVE PCR PCR PURIFICATION and PREPARATION PREPARING SLIDES PRINTING Preparing RNA: CELL CULTURE AND HARVEST Hybing the Chip: POST PROCESSING RNA ISOLATION ARRAY HYBRIDIZATION DATA ANALYSIS cdna PRODUCTION PROBE LABELING 15
Building the Chip: MASSIVE PCR PCR PURIFICATION and PREPARATION Full yeast genome = 6,500 reactions IPA precipitation +EtOH washes + 384-well format PRINTING PREPARING SLIDES Polylysine coating for adhering PCR products to glass slides The arrayer: high precision spotting device capable of printing 10,000 products in 14 hrs, with a plate change every 25 mins POST PROCESSING Chemically converting the positive polylysine surface to prevent nonspecific hybridization 16
Preparing RNA: CELL CULTURE AND HARVEST Designing experiments to profile conditions/perturbations/ mutations and carefully controlled growth conditions RNA ISOLATION RNA yield and purity are determined by system. PolyA isolation is preferable but total RNA is useable. Two RNA samples are hybridized/chip. cdna PRODUCTION Single strand synthesis or amplification of RNA can be performed. cdna production includes incorporation of Aminoallyl-dUTP. 17
Hybing the Chip: ARRAY HYBRIDIZATION Cy3 and Cy5 RNA samples are simultaneously hybridized to chip. Hybs are performed for 5-12 hours and then chips are washed. DATA ANALYSIS PROBE LABELING Two RNA samples are labelled with Cy3 or Cy5 monofunctional dyes via a chemical coupling to AA-dUTP. Samples are purified using a PCR cleanup kit. Ratio measurements are determined via quantification of 532 nm and 635 nm emission values. Data are uploaded to the appropriate database where statistical and other analyses can then be performed. 18
Affymetrix GeneChip expression array design 19
20 www.affymetrix.com
21 www.affymetrix.com
Affymetrix processing steps Quality control procedures Sample RNA isolation Gel electrophoresis, OD cdna synthesis Gel electrophoresis Biotin-labeled crna synthesis Gel electrophoresis, OD crna fragmentation Gel electrophoresis Hybridization to array Array wash and stain Array scanning Examination of the intensity of the image Image analysis Examination of chip quality indicators, and control probe sets 22
Cartoon version: Before labelling Sample 1 Sample 2 Array 1 Array 2 23
Before Hybridization Sample 1 Sample 2 Array 1 Array 2 24
After Hybridization Array 1 Array 2 25
Quantification 4 2 0 3 0 4 0 3 Array 1 Array 2 26
Adffymetrix chip image: low res. 27
Acknowledgments UCB Statistics Current and previous group members (you know who you are) CSIRO Image Analysis Group Michael Buckley Ryan Lagerstorm WEHI Many people PMCI Chuang Fong Kong Ngai Lab (Berkeley) Cynthia Duggan Jonathan Scolnick Dave Lin Vivian Peng Percy Luu Elva Diaz John Ngai LBNL Matt Callow Others Rafael Irizarry 28