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Berkeley STATISTICS 246 - Oligonucleotide microarrays for quantifying mRNA abundance

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1Oligonucleotide microarrays for quantifyingmRNA abundanceStatistics 246 Spring 2006Week 8 Lecture 22Gene expression: the central dogmaProteinmRNADNAtranscriptiontranslationCCTGAGCCAACTATTGATGAAPEPTIDECCUGAGCCAACUAUUGAUGAA3Our goal here:To measure gene expression, as indicated by mRNA abundance,on a genome-wide (transcriptome-wide) scale.Most people would prefer to measure gene expression via proteinabundance, but that is currently impossible on a genome-wide(proteome-wide) scale. However, attempts at doing so aremoving along rapidly.4Recall DNAstructureC≡G (3 H-bonds)A=T (2 H-bonds)5Hybridization This is a technique which exploits that potent feature of theDNA duplex - the sequence complementarity of the twostrands. When DNA is heated, it melts or denatures, i.e.the H-bonds break, and the two strands come apart. Remarkably, when cooled, the single-stranded DNA canthen reassemble (renature, anneal, hybridize) with perfectfidelity from the separated strands into double strandedDNA. The melting temperature of a DNA molecule is denoted byTm . This depends primarily on G+C content of thesequence, but more generally on the entire sequence ofthe molecule, salt concentration, pH and other aspects ofthe solution.6789DNA probes We now introduce the DNA probes used in the hybridizationreaction to generate the data we use to quantify mRNAaboundance in our samples. Here probe is a general term for single stranded (s.s.) DNAsequences that are complementary in the Watson-Cricksense (A-T, G-C) to their s.s. target sequences. In ahybridization reaction, complementary s.s. sequences findone another and base pair, that is, form a double-stranded(d.s.) hybrid. We say one sequence binds to the other. In the reaction, more perfect matches generally leads tostronger binding. Also, mismatches near the center generallyreduce binding more than mismatches near ends.10More on probe binding and melting tempTAGCCATCGGTAAGTACTCAATGAT Perfect (Match) pairing binds best, givingATCGGTAGCCATTCATGAGTTACTA highest melting temperature Tm; the more G-Cs, the higher the Tm.TAGCCATCGGTATGTACTCAATGAT Mismatch (probe) at one base in theATCGGTAGCCATTCATGAGTTACTA center generally has most effect on binding (among single mismatches).TAGCCATCGGTAAGTACTCAATGAT Mismatch at one base at one endCTCGGTAGCCATTCATGAGTTACTA generally has least effect on binding (among single mismatches)TAGCCATCGGTAAATACTCGATGAT Many mismatches leads to very poorATAGGTCGCCATTCATCAGTTAATA binding, low Tm.11The Affymetrix chip: 3’ probe selectionProbes are 25-mers selected from the complement of a targetmRNA sequence. Frequently probes overlap. 5-50K targetfragments are interrogated together bin probe sets of 11-20probes chosen from the 3’ end of transcripts. Affymetrixtypically uses both PM and MM probes (for why, see later).Recently, arrays with probes in all exons and elsewhere havebecome available, and these are PM only.12Probe selection rules When there is a choice to be between possible probes, theselection will me made according to a variety of criteria,weighted and combined by the chip designer. One will be theuniqueness of the probe sequence in the relevant genome:how close are the the closest alternatives? Another will be theTm, which reflects binding affinity of probe for target. A thirdmight be be the uniformity of the Tm’s across the entire set ofprobes. As we will see, the chip is hybridized at onetemperature, so all probes must perform at that temperature.A fourth might be the tendency of the probe to form secondarystructures (to be avoided). Spacing is also important: overlapis not desirable if it can be avoided, and so on. In generalhaving probes near the 3’ end increases the chance of theirbeing unique to that gene.13The Affymetrix SNP chip, cont.*****Here the probes are fixed to the chip.During the hybridization reaction,fluorescently labelled target DNA(see the s) fragments find their Watson-Crick complements and base-pair withthem.After the hybridization reaction is completed, unbound target DNAis washed off. Then the chip is stained and scanned, and afluorescent readout for every feature on it gives us our data.A larger read-out means more d.s. DNA bound to the feature, i.e.more of that probe sequence in the target DNA. (The above andmost of the figures which follow are from the Affymetrix web site.)14Affymetrix synthesizes probes onto the chip15Oligonucleotide arrays: an overview1818µµm orm orsmallersmaller101066-10-1077 copies of a specific copies of a specificoligonucleotide probe per featureoligonucleotide probe per feature Image of Hybridized Probe ArrayImage of Hybridized Probe Array>450,000 different probes>450,000 different probesSingle stranded, Single stranded, labeled RNA targetlabeled RNA targetOligonucleotide probeOligonucleotide probe*****1.28cm1.28cmGeneChipGeneChip Probe ArrayProbe ArrayHybridized Probe CellHybridized Probe Cell*16GeneChip eukaryotic expression assay171819RNA isolationcDNA synthesiscRNA fragmentationHybridization to arrayArray wash and stainArray scanningImage analysisProcessing step QA/QC procedures Gel electrophoresis, OD Gel electrophoresis Examination of the intensity of imageExamination of chip quality indicators,and control probe sets Gel electrophoresis, OD Gel electrophoresisBiotin-labeled cRNA synthesis20Obtaining the dataRNA samples are prepared, labeled, hybridized witharrays, and stained as just describedArrays are scanned and the resulting image analyzed(we omit the details)Approximately 30-50 pixels per probe cell (now fewer)are summarized by their 75th %tile, after removal ofouter perimeter pixels. This is the probe cell’sintensity.Of interest is to find a way to combine probe cellintensities for a given gene to produce an index ofexpression – an indicator of abundance of thecorresponding target mRNA. We discuss this later.21Cartoon version of the process(courtesy of Rafael Irizarry)followed by some real images22After fragmenting, before target labellingProbe sequences fixed to chipTarget fragments23After labelling, before hybridizationLabelled s.s. target mRNA(actually cRNA)24After hybridization and wash25Ideal quantification4 2 0 326Actual quantification: Imaging by a scanner27Chip dat file – checkered board – oligo B228Chip dat file – checkered


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Berkeley STATISTICS 246 - Oligonucleotide microarrays for quantifying mRNA abundance

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