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Microarrays, RNAseq And Functional Genomics CPSC265 Matt Hudson Microarray Technology • Relatively young technology – • Already mostly obsolete, though. • Usually used like a Northern blot – can determine the amount of mRNA for a particular gene • Except – a Northern blot measures one gene at a time • A microarray can measure every gene in the genome, simultaneously Recent! History • 1994. First microarrays developed by Ron Davis and Pat Brown at Stanford. • 1997-1999. Practical microarrays become available for yeast, humans and plants Why analyze so many genes? • Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function. • To find genes involved in a particular process, we can look for mRNAs “up-regulated” during that process. • For example, we can look at genes up-regulated in human cells in response to cancer-causing mutations, or look at genes in a crop plant responding to drought. • Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways Two Main Types of Microarray Oligonucleotide, photolithographic arrays “Gene Chips” Miniaturized, high density arrays of oligos (Affymetrix Inc., Nimblegen, Inc.) Printed cDNA or Oligonucleotide Arrays Robotically spotted cDNAs or Oligonucleotides • Printed on Nylon, Plastic or Glass surface • Can be made in any lab with a robot • Several robots in ERML • Can also buy printed arrays commercially The original idea A microarray of thousands of genes on a glass slide Each “spot” is one gene, like a probe in a Northern blot. This time, the probes are fixed, and the target genes move about. Glass slide microarray summary The process Building the chip: MASSIVE PCR PCR PURIFICATION and PREPARATION PREPARING SLIDES RNA preparation: CELL CULTURE AND HARVEST PRINTING Hybing the chip: POST PROCESSING ARRAY HYBRIDIZATION RNA ISOLATION DATA ANALYSIS cDNA PRODUCTION PROBE LABELING steel Robotically printed arrays spotting pin chemically modified slides 384 well source plate 1 nanolitre spots 90-120 um diameter Physical Spotting Labelling RNA for Glass slides Reverse Transcriptase Reverse transcription mRNA Cy3 label (control) mRNA (treated) Cy5 label cDNA Cy3 labelled cDNA Cy5 labelled Hybridization Binding of cDNA target samples to cDNA probes on the slide Hybridize for 5-12 hours Northern blot vs. Microarray • In Northern blotting, the whole mRNA of the organism is on the membrane. The labelled “probe” lights up a band – one gene • In a microarray, the whole genome is printed on a slide, one “probe” spot per gene. Mixed, labelled cDNA, made from mRNA from the organism, is added. Each probe lights up green or red according to whether it is more or less abundant between the control and the treated mRNA. Hybridization chamber 3XSSC HYB CHAMBER ARRAY LIFTERSLIP SLIDE LABEL SLIDE LABEL • Humidity • Temperature • Formamide (Lowers the Tm) Expression profiling with DNA microarrays cDNA “B” Cy3 labeled cDNA “A” Cy5 labeled Laser 1 Hybridization Laser 2 Scanning + Analysis Image Capture Image analysis GenePix Spotted cDNA microarrays Advantages • Lower price and flexibility • Can be printed in well equipped lab • Simultaneous comparison of two related biological samples (tumor versus normal, treated versus untreated cells) Disadvantages • Needs sequence verification • Measures the relative level of expression between 2 samples Affymetrix Microarrays • One chip per sample • Made by photolithography • ~500,000 25 base probes …unlike Glass Slide Microarrays •Made by a spotting robot •~30,000 50-500 base probes •Involves two dyes/one chip •Control and experiment on same chip Affymetrix GeneChip Miniaturized, high density arrays of oligos 1.28-cm by 1.28-cm (409,000 oligos) Manufacturing Process Solid-phase chemical synthesis and Photolithographic fabrication techniques employed in semiconductor industry Selection of Expression Probes Set of oligos to be synthesized is defined, based on its ability to hybridize to the target genes of interest 5’ 3’ Sequence Probes Perfect Match Mismatch Chip Computer algorithms are used to design photolithographic masks for use in manufacturing Photolithographic Synthesis Manufacturing Process Probe arrays are manufactured by light-directed chemical synthesis process which enables the synthesis of hundreds of thousands of discrete compounds in precise locations Lamp Mask Chip Affymetrix Wafer and Chip Format 20 - 50 µm 50… 11µm Millions of identical oligonucleotides per feature 49 - 400 chips/wafer 1.28cm up to ~ 400,000 “features” / chip Labelling RNA for Affymetrix Reverse Transcriptase Reverse transcription mRNA cDNA in vitro transcription cRNA Transcription Biotin labelled nucleotides Target Preparation B Biotin-labeled transcripts B B B B Fragment (heat, Mg2+) B B cDNA Fragmented cRNA Wash & Stain Scan AAAA mRNA B Hybridize (16 hours) ® GeneChip Expression Analysis Hybridization and Staining Array Hybridized Array cRNA Target Streptravidinphycoerythrin conjugate Example: Comparing a mutant cell line with a wild type line. Instrumentation Affymetrix GeneChip System 3000-7G Scanner 450 Fluidic Station Microarray data analysis This is now a very important branch of statistics It is unusual to do thousands of experiments at once. Statistical methods didn’t exist to analyse microarrays. Now they are being rapidly developed. Normal vs. Normal Normal vs. Tumor Lung Tumor: Up-Regulated Lung Tumor: Down-Regulated Microarray Technology - Applications • Gene Discovery– Assigning function to sequence – Finding genes involved in a particular process – Discovery of disease genes and drug targets • Genotyping – – – – SNPs Genetic mapping (Humans, plants) Patient stratification (pharmacogenomics) Adverse drug effects (ADE) • Microbial ID Why it is becoming obsolete • In a word, RNAseq • RNAseq uses DNA sequencing to do the same thing. • Rather than an array, you just sequence millions of mRNA fragments, then figure out what genes they are from Why RNAseq only just caught on • It’s been around for a long time, called things like SAGE and MPSS. • But they were expensive and arrays were cheap. Now, sequencing is as cheap as arrays • Also, you need a fully sequenced reference genome for the computer analysis. What RNAseq / arrays can’t do • Tell you anything about protein levels • Tell you anything about post-translational modification of proteins • Tell you anything about the structure of proteins • Predict the phenotype of a genetic mutant Proteomics • A high througput approach to learning about all the proteins in a cell • As microarrays are to a Northern blot, proteomics is to a Western blot • Two main approaches – • 2D gels + MS • Protein microarrays Protein separation: 2-dimensional gel electrophoresis 1st dimension Separation by charge (isoelectric focussing) pI pH 3 pH 10 2nd dimension Separation by molecular weight (SDS-PAGE) kDa Susan Liddel Proteins extracted from cow ovarian follicle granulosa cells separated on a broad range IPG strip (pH3-10) followed by a 12.5% polyacrylamide gel, silver stained 3.5 9.0 150 100 75 50 37 25 20 Susan Liddel Mass Spectrometry FT-MS can tell you 10-20 residues of sequence, but only from a purified protein Robots pick spots from 2-D gel, load into MS Also, 2-D and 3-D LC Array-based protein interaction detection Protein microarrays The future of microarrays: •Still looking good, in areas other than research •Used by pharmaceutical companies, medical diagnostics, etc. •In the future, just like silicon chips, likely to get cheaper, faster and more powerful •It may not be long before they are routinely used to diagnose disease The future of proteomics: • Many people will tell you proteomics IS the future of biology • If they can get it to work as well as microarrays, they will be right • The problem is, every protein has different chemistry, while all mRNAs are closely comparable • At the moment, proteomics is a hot field, but few major biological discoveries have been made with proteomics – many have been made with microarrays