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Gene Expression Data Analyses (1) Trupti Joshi Computer Science Department 317 Engineering Building North E-mail: [email protected] 573-884-3528(O) Lecture Schedule for Gene Expression Analyses Concept of microarray and experimental design for DNA microarray (9/6/05) Data transformation and normalization for DNA microarray (9/8/05) Statistical analysis for DNA microarray and Software comparison (9/13/05) Clustering Techniques for DNA microarray (Dr. Dong Xu 9/15/05) Lecture Outline Central Dogma of Molecular Biology Introduction to Gene Expression and Microarray Experimental Design Lecture Outline Central Dogma of Molecular Biology Introduction to Gene Expression and Microarray Experimental Design Central Dogma of Molecular Biology Gene Expression mRNA level Protein level Lecture Outline Central Dogma of Molecular Biology Introduction to Gene Expression and Microarray Experimental Design Introduction: Gene Expression Same DNA in all cells, but only a few percent common genes expressed (house-keeping genes). A few examples: (1) Specialized cell: over-represented hemoglobin in blood cells. (2) Different stages of life cycle: hemoglobins before and after birth, caterpillar and butterfly. (3) Different environments: microbial in nutrient poor or rich environment. (4) Diversity of life. Microarray is about gene expression. All information about living being is coded in DNA as a set of genes. Each gene contains structural information about protein sequence and regulatory information about protein expression. Intermediate step between gene and protein is mRNA. The concentration of mRNA is measured by microarray. Problem RNA levels and protein levels are not always directly correlated. No mRNA no protein; Relation is not simple and not universal. Functional genomics fill the gap between gene expression and organism function. The meaning of life is hidden in gene expression value but it is not easy to get it out. Eucaryote Gene Expression Control nucleus DNA inactive mRNA mRNA degradation control Primary RNA transcript transcriptional control cytosol mRNA RNA processing control Microarray mRNA Mass-spec protein RNA transport control mRNA translation control protein nucleus membrane protein activity control inactive protein Principle of DNA Microarray Complimentary hybridization is the basis of RNA measurement. Base-pairing rules DNA: A-T and G-C RNA: A-U, G-C, G-U A--T G--C T--A C--G Microarray Technology Macroarray: sample spot sizes >= 300 microns Microarray: typically < 200 microns biochip, DNA chip, DNA microarray, gene array, genome array, gene chip Initial Ideas of DNA Microarray Immunoassay Ekins, R. and F. W. Chu. Microarrays: their origins and applications. Trends in Biotech. 17: 217-218 Application of DNA Microarray Technology Gene discovery Biological mechanisms (gene regulatory network, etc.) Disease diagnosis (cancer, infectious disease, etc.) Drug discovery: Pharmacogenomics Toxicological research: Toxicogenomics Microbial diversity in the environment … Increasing Microarray Applications Advantages and Disadvantages of Micoarray Advantages: High-throughput Analyze gene expressions of different cells or from cells under different condition simultaneously Disadvantages: High noise Relatively high cost Categories of DNA Microarray Probe based cDNA microarray: cDNA (500~5,000 bases) as probe. 10,00020,000 spots/slide. Oligo microarray (Affimetrix Microarray): oligonucleotide (20~80mer oligos) as probe. 200,000-500,000 spots/slide. Dye based Double label. For example, Cy3 and Cy5. One sample is labeled with a “green” dye and the other with “red”. Relative fluorescent intensity of red and green from the same spot. Single label. All samples are labeled with one color. Absolute fluorescent intensity between different slides. Does not control for the amount of DNA in each spot. Chips Typically a glass slide with cDNA or oligo Printed by robot or synthesized by photo-lithography. Typical arrays are 25x75 mm. Contains up to 500,000 probed gene fragments. Probe Layout on Chips Positive control Genome DNA House keeping genes Negative control Spots with cDNA from very different species Blank spots Spots with buffer Samples Technical replicates Microarray Procedures Experimental Design RNA extraction cDNA prepration Data interpretation Statistical analysis Data transformation and Normalization cDNA labeling Sample mixing Hybridization Image Analysis Scanning Molecular Interaction on microarray 1 molecule per square angstroms Large molecules are easily to be folded by themselves Short targets are better than large targets to interact with tethered oligos Ideally, target and probe should have the same length Molecules interaction are dynamic Competitive hybridization Lecture Outline Central Dogma of Molecular Biology Introduction to Gene Expression and Microarray Experimental Design Experimental Protocol A. Synthesis of cDNA Synthesis of the second strand DNA B. Labeling C. Hybridization D. Scanning Rational for Experimental Design Scientific constrains: Scientific aims and their priorities Physical constrains: Number of slides Amount of mRNA Goal of an optimal design: Minimize costs from money, time Maximize the useful information Issues for Experimental Design Scientific Specific questions and their priorities. Practical (logistic) Types of mRNA samples: reference, control, treatment. Amount of material available (mRNA, slides, dyes). Other factors The experimental process before hybridization: sample isolation, mRNA extraction, amplification, and labeling. Controls planned: positive, negative, ratio, and so on. Verification method: northern blot, reverse transcriptase (RT)-PCR, in situ hybridization, and so on. Variability and Replicates Gene expression level for one gene in different slides may not be the same Replicates: Technical replicates: the target mRNA is from the same pool (RNA extraction) Reduce variability Biological replicates: the target mRNA is from different individual extraction. Obtain averages of independent data Validate generalizations of conclusions Variation within technical replicates are smaller than that within Biological replicates Importance of Replicates Graphical Representation of Design Cy3: green Cy5: red Cy3+Cy5: blue Use directed graphs Node: sample Edge: hybridization, use Cy3 Cy5 Weight: replicates Direct & Indirect Comparison Compared objectives: T and C Directive design: TC are on the same slide Indirect design: TR and CR are on the same slides, respectively. But T and C are on different slides Variance & Std Deviation Variance The most common statistical measure of variability of a random quantity or random sample about its mean. Its scale is the square of the scale of the random quantity or sample. Standard Deviation Standard deviation is the square root of the variance. It measures the spread of a set of observations. The larger the standard deviation is, the more spread out the observations are. Variance for Indirect Design For sample T and C: log 2 T α and β are means of log intensities across slides for a typical gene. Differential Expression Direct design ^ D 1 / 2(log 2 (T / C ) log 2 (T ' / C ' )) var( D ) / 2 2 Indirect design ^ D log 2 (T / R ) log 2 (C / R' ) var( D ) 2 2 log 2 C Dye-swapped Replication Two sets of replications Dye-swapped replications Two hybridizations for two mRNA samples are on the two slides, but dye swapped. For example, Cy3 for A and Cy5 for the first hybridization (slide 1), then C5 for A and Cy3 for the second hybridization (slide 2). Advantage: reduce systematic bias (e.g. dye bias) Reference Design It may not be feasible to perform direct design when experimental conditions are more than 3. Factors in the design Single Two factor factors Multiple factors Single Factor Experiments Time-course Experiments 2x2 factorial experiments Lecture Outline Central Dogma of Molecular Biology Introduction to Microarray Application Advantage vs. Disadvantage Chips Microarray procedure Experimental design Rational Variability and Replication Graphical representation Direct comparison and Indirect comparison Dye swap Reference design Single-factor design Multifactorial design Reading Assignments Suggested reading: Yang, YH and T. Speed. 2002. Design issues for cDNA microarray experiments. Nature Reviews, 3: 579-588. Statistical analysis of gene expression microarray data. Chapter 2. pp. 35-92. Chapman&Hall/CRC Press, 2003.