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 9 The cDNA and short 25 bp oligo technologies in brief Long 60 75 bp oligo arrays are more like the cDNA ones 10 excitation cDNA clones probes cDNA arrays in summary PCR product amplification purification printing laser 2 scanning laser 1 emission mRNA target overlay images and normalise 0 1nl spot microarray Hybridise target to microarray analysis 11 Affymetrix GeneChips in summary details slightly out of date GeneChip Probe Array Hybridized Probe Cell Single stranded labeled RNA target Oligonucleotide probe 24 24 m 1 28cm Millions of copies of a specific oligonucleotide probe synthesized in situ grown 200 000 different complementary probes Image of Hybridized Probe Array 12 Compliments of D Gerhold 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 cDNA arrays the process Building the Chip MASSIVE PCR PCR PURIFICATION and PREPARATION PREPARING SLI DES PRINTING Preparing RNA CELL CULTURE AND HARVEST Hybing the Chip POST PROCESSING ARRAY HYBRIDIZATION RNA ISOLATION DATA ANALYSIS cDNA PRODUCTION PROBE LABELING 15 Building the Chip MASSIVE PCR Full yeast genome 6 500 reactions PCR PURIFICATION and PREPARATION IPA precipitation EtOH washes 384 well format PRINTING PREPARING SLI DES 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 cDNA synthesis Biotin labeled cRNA synthesis cRNA fragmentation Gel electrophoresis OD Gel electrophoresis Gel electrophoresis OD 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 Array 1 Sample 2 Array 2 23 Before Hybridization Sample 1 Array 1 Sample 2
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