Microarray Technology

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1 Microarrays And Functional Genomics CPSC265 Matt Hudson Microarray Technology Relatively young technology 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 First microarrays developed by Ron Davis and Pat Brown at Stanford Practical microarrays become available for yeast, humans and plants 1

2 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. 2

3 Glass slide microarray summary The process Building the chip: MASSIVE PCR PCR PURIFICATION and PREPARATION PREPARING SLIDES PRINTING RNA preparation: CELL CULTURE AND HARVEST Hybing the chip: POST PROCESSING RNA ISOLATION ARRAY HYBRIDIZATION cdna PRODUCTION PROBE LABELING DATA ANALYSIS steel spotting pin Robotically printed arrays chemically modified slides 1 nanolitre spots um diameter 384 well source plate 3

4 Physical Spotting Labelling RNA for Glass slides mrna (control) Reverse transcription Cy3 label Reverse Transcriptase cdna Cy3 labelled mrna (treated) Cy5 label cdna Cy5 labelled 4

5 Hybridization Binding of cdna target samples to cdna probes on the slide cover slip 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 LABEL SLIDE SLIDE LABEL Humidity Temperature Formamide (Lowers the Tm) 5

6 Expression profiling with DNA microarrays cdna A Cy5 labeled cdna B Cy3 labeled Laser 1 Laser 2 Hybridization 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 6

7 Affymetrix Microarrays One chip per sample Made by photolithography ~500, base probes unlike Glass Slide Microarrays Made by a spotting robot ~30, 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 7

8 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 µm 50 11µm Millions of identical oligonucleotides per feature chips/wafer 1.28cm up to ~ 400,000 features / chip 8

9 Labelling RNA for Affymetrix Reverse transcription Reverse Transcriptase mrna cdna in vitro transcription crna Transcription Biotin labelled nucleotides Target Preparation Biotin-labeled transcripts B B B B Fragment (heat, Mg 2+ ) B B B B cdna Fragmented crna Wash & Stain Scan AAAA mrna Hybridize (16 hours) GeneChip Expression Analysis Hybridization and Staining Array Hybridized Array crna Target Streptravidinphycoerythrin conjugate 9

10 Example: Comparing a mutant cell line with a wild type line. Instrumentation Affymetrix GeneChip System G 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. 10

11 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 11

12 What DNA microarrays 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 12

13 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 Susan Liddel Mass Spectrometry FT-MS can tell you 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 13

14 Protein microarrays The future of microarrays: Still looking good Used by most pharmaceutical companies, almost all University biology departments 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 14

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