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