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Microarray_Introduction Methods for the Study of Gene Expression Gabriela Salinas-Riester November 2012 www.microarrays.med.uni-goettingen.de Transcriptome Analysis Labor Microarray and Deep Sequencing Core Facility UMG Methods for the Study of Gene Expression Microarray_Introduction Outline ➢ From genotype to phenotype: the transcriptome ➢ Types of RNAs and their functions ➢ Methods for Study gene expression ➢ Single transcripts ➢ Multiple transcripts simultaneously: NanoString, Microarrays, NGS ➢ What are microarrays and how do they work? ➢ Agilent platform ➢ Experimental Workflow ➢ Sample Quality control ➢ Agilent Microarray Workflow ➢ Data Analysis Gabriela Salinas-Riester Methods for the Study ofmedicine_2011 Gene Expression 2/22 Linking genome-wide analysis to genomic Microarray_Introduction Gabriela Salinas-Riester Linking genome-wide Methods analysis for the to genomic Study of medicine_2011 Gene Expression 2/22 Microarray_Introduction Gabriela Salinas-Riester Linking genome-wide Methods analysis for the to genomic Study of medicine_2011 Gene Expression 2/22 Microarray_Introduction Types of RNAs and their functions Long RNAs Function mRNA (0.4-10 kb) Coding genes lincRNA (>200 bp) Regulating basal transcription machinery rRNA (1898-3898 bp) protein synthesis (28S and 18S) Small RNAs Function snRNA RNA splicing/Regulation of transcription snoRNA (60-300 bp) RNA modification ● ● C/D box snoRNAs H/ACA box snoRNAs miRNA (19-24 bp) Translational repression, mRNA cleavage piRNAs (26-31nt) Piwi-interacting RNA Transposone repression/DNA methylation tiRNAs (17-18nt) tini RNAs Associated with TSS in animals tRNA (73-95 bp) Translation rRNA (5.7 S and 5.0 S) protein synthesis mt-tRNA Mitochondrial Translation siRNA (small interfering RNA) RNA silencing gRNA (guide RNA) RNA editing TSSa-RNAs (20-90 bp) Maintenance of transcription? Methods for the Study of Gene Expression Microarray_Introduction Methods for the Study of Gene Expression PCR 1986 Northern Blot Microarrays 2000 RPA qRT-PCR 1953 NGS 2008 Single Transcripts: ● Northern blot ● RNAse protection assay ● Reverse Transcription (RT)-PCR ● Real-time PCR (qPCR) ● In-situ-hybridization Multiple Transcripts Simultaneously: ● Dot-Blot analysis ● Differential Display, SAGE ● Subtractive hybridization (1996) ● DNA Microarrays (1999) ● NanoString ● Second generation Sequencing (NGS) Methods for the Study of Gene Expression Microarray_Introduction Methods for the Study of Gene Expression ● Northern blot ● Subtractive hybridization Methods for the Study of Gene Expression Microarray_Introduction What are microarrays and how do they work? Methods for the Study of Gene Expression Microarray_Introduction Agilent Platform v Methods for the Study of Gene Expression Microarray_Introduction Advantages and Disadvantages of microarray techniques Advantages: ● Concurrent detection of thousands of genes ● Result is a global answer ● Experiments are fast and easy to perform ● The technique is cost effective Disadvantages: ● Expression differences are relativ ● Crosshybridization is possible ● Data has to be validated by other methods (eg. qPCR) ● Weak expressed genes may not be detected ● Changes in mRNA expression pattern may not effect protein abundance Methods for the Study of Gene Expression Microarray_Introduction Agilent Microarray Workflow Day 1 Day 2 Data Analysis Methods for the Study of Gene Expression Microarray_Introduction Experimental Workflow 6.Scan/Image (Raw Data) 1.Sample preparation v 5.Wash & Stain Hybridization 2.Quality Control 3.Sample labeling 4.Quality Control and Amplification Methods for the Study of Gene Expression Microarray_Introduction Sample Quality Control Total RNA acRNA NanoChip (Agilent) Fragmented acRNA Methods for the Study of Gene Expression Microarray_Introduction Bioinformatics ➢ The bioinformatic analysis include: ➢ Overview of image processing ➢ Logarithmic transformation ➢ Data Normalization ➢ Data Reduction ➢ Hierarchical Clustering ➢ Principal Component Analysis (PCA) ➢ Statistical Analysis ➢ List of candidates ➢ Data Visualization (Heatmaps) Methods for the Study of Gene Expression Microarray_Introduction Data Normalization Methods for the Study of Gene Expression Microarray_Introduction Hierarchical Clustering Methods for the Study of Gene Expression Microarray_Introduction Principal Component Analysis Methods for the Study of Gene Expression Microarray_Introduction Heat Map Analysis for a specific pathway Methods for the Study of Gene Expression