Microarrays Wednesday March 1 2006 Dr Tim Hughes CCBR 160 College St Room 1302 t hughes utoronto ca Outline Microarray experiments Normalization Different types of microarrays Other applications besides expression profiling Clustering and interpretation Suggested reading Eisen et al 1998 HARTIGAN J A Clustering Algorithms Wiley New York and London 1975 My understanding is that it is no longer in print but is available on CD Jain et al ACM Computing Surveys 31 3 1999 Data Clustering a review http www amk alt neustadt at diplom papers Clustering p264 jain pdf Hegde et al A concise guide to cDNA microarray analysis Biotechniques 2000 Sep 29 3 548 50 552 4 556 Sherlock G Analysis of large scale gene expression data Curr Opin Immunol 2000 Apr 12 2 201 5 Nucleic Acid Hybridization www accessexcellence org AB GG nucleic html Microarray expression profiling by 2 color assay cDNA arrays Array PCR products 6250 yeast ORFs hybridized cDNAs green control red experiment Schena et al 1995 cDNA microarrays are essentially dot blots on glass slides 0 45 mm http arrayit com Products Printing Stealth stealth html This slide was made with 16 pins 4 5 mm pin spacing matches 384 well plates 16 x 24 Done with robotics Slides usually coated with poly lysine Spots are usually 100 150 microns Spot spacing is usually 200 300 microns Slides are 25 x 75 mm Easy to deposit 20K spots slide Common ways to label nucleic acids Random priming of double stranded DNA Reaction contains labelled nucleotides Direct labelling fluors only Amplification AAAAAAAA AAAAAAAA Poly T primed cDNA synthesis TTTTTTTTTT T7 promoter AAAAAAAA Reaction contains labelled nucleotides second strand synthesis AAAAAAAA T7 promoter TTTTTTTTTT T7 promoter AAAAAAAA TTTTTTTTTT T7 reaction contains labelled nucleotides Typical use of cDNA microarrays Internal normalization using two colors treatment control drug mutation x x y y z z x x x y y cDNA pools z x y z up down unchanged not present 532 nm laser green excites Cy3 Cy3 detected with an emission filter that passes 557 592 nm Excitation 635 nm red excites Cy5 Cy5 detected with an emission filter that passes 650 690 nm Both are detected by a photomultiplier tube Cy3 NHS Ester Emission Cy5 NHS Ester http www jacksonimmuno com 2001site home catalog f cy3 5 htm http www ope tech com doc Cy5 structure htm The primary data two grayscale TIFF files Cy3 channel green Cy5 channel red http www axon com GN GenePix4000 html Image processing and normalization what is microarray data Microarray data is summary information from image files that come out of the scanner Image processing line up grids flag bad spots quantitate Looking at data from a single experiment 2 P value 0 01 1 5 1 Log10 Expression Ratio log10 ratio 3 AT vs No drug Slides 11120c01 11121c01 2 1 0 5 0 0 0 5 1 1 1 5 2 2 2 2 1 0 1 0 5 0 0 5 Log10 Intensity 1 1 1 5 2 2 Slides 11857c01 11858c01 2 P value 0 01 1 5 1 Log10 Expression Ratio log10 ratio wild type vs wild type 2 1 5 1 0 5 0 0 0 5 1 1 1 5 2 2 2 2 1 5 1 1 0 0 5 0 0 5 Log10 Intensity 1 1 1 5 2 2 log10 average intensity Lowess smoothing The names lowess and loess are derived from the term locally weighted scatter plot smooth as both methods use locally weighted linear regression to smooth data http www mathworks com access helpdesk help toolbox curvefit ch data7 html Find spots Manual edit Quantitate Normalize Lowess smoothing Locally weighted scatterplot smoothing Confirm spots outside envelope Save data images spot map Selected tricks for processing and normalization 1 High pass spatial detrending See O Shai Q Morris and B J Frey 2003 Spatial Bias Removal in Microarray Images University of Toronto Technical Report PSI 200321 http www psi utoronto ca ofer detrendingReport pdf 2 VSN Variance Stabilizing Normalization See Huber W Von Heydebreck A Sultmann H Poustka A Vingron M Variance stabilization applied to microarray data calibration and to the quantification of differential expression Bioinformatics 18 Suppl 1 S96 S104 2002 Q Morris B Frey O Shai Other types of arrays Photolithographic arrays Affymetrix Building up oligonucleotides on a surface http www affymetrix com technology manufacturing index affx Photolithographic arrays Affymetrix Arrays are typically 25 mers with mismatch control for specificity aka GeneChip Photolithographic arrays Affymetrix Advantages Density is limited essentially by the 5 micron resolution of scanners solution larger arrays Well developed protocols Industry standard largely self driven Disadvantages Not all probes work well Affymetrix has evolved a complicated system to compensate for this but even believers use at least four probes per gene and usually more Single color Sample preparation typically requires amplification Single supplier historically intellectual property issues i e comparisons Ink jet arrays Agilent G A A G T C C C T G G G A G A 25 000 oligos 1 x 3 inches Sequence completely flexible 60 mers Hughes TR et al Expression profiling using microarrays fabricated by an ink jet oligonucleotide synthesizer Nat Biotechnol 2001 Apr 19 4 342 7 Ink jet arrays generally agree with spotted cDNA arrays r 0 97 HXT4 HXT3 cDNA array Spo vs SC r 0 96 single oligo multiple oligos Yeast IJS array 8 oligos per gene HXT1 cDNA array Ink jet arrays Agilent Advantages User specified sequences no questions asked Sensitivity and specificity are defined and exceed requirement for most expression profiling applications no amplification required Virtually every 60 mer is functional Data correlates well with spotted cDNA arrays Disadvantages Density currently limited to 45 000 spots per array Single supplier although a protocol is in press for making your own synthesizer Maskless arrays Nimblegen http www nimblegen com technology manufacture html Maskless arrays Nimblegen Advantages User specified sequences Density is limited essentially by the 5 micron resolution of scanners Disadvantages New to arena Performance in initial publication Nuwaysir et al Genome Research 2002 suggests that sensitivity and specificity may be lower than that of Agilent arrays Single supplier although all the parts are there for academics to build one Possible IP issues Hybs are done in Iceland to bypass Affy IP Nimblegen web site boasts of new partnership with Affymetrix Applications beyond expression profiling DNA copy number Genotyping Protein DNA associations Molecular Barcoding Protein arrays Transformation arrays Identifying DNA binding sites Science 2000 Dec 22 290 5500 23069