Microarray Simultaneously determining the abundance of multiple(100s-10,000s) transcripts
Gene expression assays DNA microarrays rely on the hybridization properties of nucleic acids to monitor transcript or RNA abundance on a genome-wide scale in different types of cells. The main types of gene expression assays: -High-density nylon membrane arrays (macroarrays) -Short oligonucleotide arrays (Affymetrix) -Long oligonucleotide arrays (Agilent) -cDNA arrays -genome arrays
Uses for Microarray Determination of transcript (mRNA) abundance Identification of genes (hypothetical, microRNA) Diagnostics (disease, stress, chemicals, or pharmaceuticals) Distinguish signaling pathways Determination of RNA decay rates
Microarray Experiments; different approaches to compare mRNA accumulation Different tissues, same organism (brain versus liver) Same tissue, same organism (treatment versus control, tumor versus normal) Same tissue, different organisms (wild-type versus knockout or mutant) Time course experiments (effect of treatment, development) Other special designs (e.g. to detect spatial patterns)
Question Sample Preparation Data Analysis Microarray Hybridization Microarray Detection Overview of the process
Make Chip Genomic DNA or cDNA PCR fragment purification and preparation Prepare RNA and label cDNA Grow and/or treat sample Isolate RNA cDNA synthesis and labeling Hybridization Print array Data analysis
Genomic versus complementary DNA Amplification of genomic DNA with the Polymerase Chain Reaction (PCR) Amplification of complementary DNA (cDNA) with the Polymerase Chain Reaction (PCR) AAAAA TTTTTT mRNA Oligo-T primer dNTPs Reverse transcriptase (RT) RT
Denaturation Step 94 o C for 1 min. Annealing Step o C for 30 sec. Extension Step 72 o C for 2-5 min cycles PCR Taq
PCR Exponential increase in target DNA Target DNA Number of cycles Copies of target DNA
Building a cDNA chip Arrayed Library (96 or 384-well plates of bacterial glycerol stocks) PCR amplification of target DNA (cDNA or portion of genomic DNA) Consolidate into plates Spot as microarray on glass slides
Make Chip Glass slide
Microarray Analysis
Label cDNA Single stranded cDNA (antisense) Cy3 labeled Reverse transcribe and label Reverse transcriptase Oligo-T primer dNTPs (Cy3 labeled) Isolate polyA + RNA from control (wild-type or non-treated) Single stranded cDNA (antisense) Cy5 labeled Isolate polyA + RNA from treatment, mutant, or transgenic organism Reverse transcribe and label Reverse transcriptase Oligo-T primer dNTPs (Cy5 labeled) AAAAAAAAAAA TTTTTT
Hybridization of nucleic acids DNA fixed to a matrix matrix = membrane (nylon or nitrocellulose) or glass 5’-GGGCCCATGCATTTAAGCATGGCATGCAT-3’ Matrix III III III III III III II II III III II II II II II II III III II II III III III II II III III II II 3’-CCCGGGTACGTAAATTCGTACCCTACGTA-5’ III III III III III III II II III III II II II II II II III III II II III III III II II III III II II 3’-CCCGGGTACGTAAATTCGTACCCTACGTA-5’
Expression profiling with DNA microarrays cDNA “control” Cy3 labeled cDNA “treament” Cy5 labeled Hybridization Scanning Laser 1 Laser 2 + AnalysisImage Capture
LABEL 3XSSC HYB CHAMBER ARRAY SLIDE LIFTERSLIP SLIDE LABEL Humidity Temperature Formamide Hybridization chamber
Microarray Analysis Data storage and retrieval Filtering Normalization Analysis
Image analysis
Quantification Cy5 at 635Cy3 at 532 Overlay images
Expression profiling with DNA microarrays Data acquistion HYBRIDIZATION Spot #1 Spot #3 Spot #2 Spot #4 Spot #6 Spot #5 Relative intensity Relative intensity cDNA “control” Cy3 labeled Output cDNA “treament” Cy5 labeled
Sample preparation
Microarray Analysis Data storage and retrieval Filtering Normalization Analysis
Microarray Analysis Data Quality Irregular size or shape Irregular placement Low intensity Saturation Spot variance Background variance indistinguishablesaturated bad print artifactmiss alignment
Replicate spots
Reverse Dye-Label Reverse-dye label
Microarray Analysis Hierarchical Clustering
Get a Global Perspective; Plot Data on a Metabolic Chart Data set from an experiment with Arabidopsis dataset_aracyc.txt Metabolic pathway analysis
Microarray Analysis Tutorial Cluster and TreeView Free download from Data set for cluster and treeview aset.txt Tutorial as part of a course (outline, test,data and links to free downloads Tutorial outline ay_tutorial_WS2004.pdf