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Unformatted text preview: 1 1 Statistical Design and Analysis of Microarray Experiments 1/17/2008 Peng Liu 2 Microarray Technology Â¡ How to measure expressions of thousands of genes simultaneously? Â¡ Two types of fabrication for microarray: spotted microarray (cDNA microarray, 500-5k bp long) Oligonucleotide microarray (Affymetrix microarray/GeneChip Â®, 20~80-mer oligos) 3 complementary DNA (cDNA) Â¡ cDNA is a strand of DNA that is complementary to part of an mRNA sequence. Â¡ cDNA can be formed by extracting mRNA and then using mRNA as a template for formation of cDNA Â¡ cDNA sequences can be copied rapidly using PCR (polymerase chain reaction). Â¡ These sequences can be spotted on glass slides to serves as microarray probes. Â¡ Sequence length varies from a few hundred bases to a thousand or so. ...CCUGAUAGAUGG... mRNA ...GGACTATCTACC... cDNA 4 cDNA Microarrays Â¡ Glass slides or similar supports containing cDNA sequences that serve as probes for measuring mRNA levels in target samples Â¡ cDNAs are arrayed on each slide in a grid of spots. Â¡ Each spot contains thousands of copies of a sequence that matches a segment of a geneâ€™s coding sequence. Â¡ A sequence and its complement are present in the same spot. 5 cDNA Microarray (continued) Â¡ Different spots typically represent different genes, but some genes may be represented by multiple spots Â¡ The spotted sequences are known (or can be determined) and their locations on the array are known. Â¡ The sequence locations do not change from slide to slide. Â¡ A single slide typically contains thousands of spots. 6 cDNA microarray slide 2 cDNA microarray slide 1 TTCCAG... TTCCAG... TTCCAG... ... GATATG... GATATG... GATATG... ... Each spot contains many copies of a sequence along with its complement (not shown). spot for gene 201 spot for gene 576 TTCCAG... TTCCAG... TTCCAG... ... GATATG... GATATG... GATATG... ... spot for gene 201 spot for gene 576 2 7 Spotting cDNA Probes on Microarrays Â¡ Solutions containing probes are transferred from a plate to a microarray slide by a robotic arrayer. Â¡ The robot picks up a small amount of solution containing a probe by dipping a pin into a well on a plate. Â¡ The robot then deposits a small drop of the solution on the microarray slide by touching the pin onto the slide. Â¡ The pin is washed and the process is repeated for a different probe. Â¡ Most arrayers use several pins so that multiple probes are spotted simultaneously on a slide. Â¡ Most arrayers print multiple slides together so that probes are deposited on several slides prior to washing. 8 The PixSys 5500 Arraying Robot (Cartesian Technologies) Vacuum wash station The print head holds up to 32 pins in a 8x4 format Vacuum hold-down platform (50 slide capacity) Robotic arm 9 Plate with wells containing probes microarray slides vacuum wash station Cartoon of Printing Process (side view from the table top) 10 Spotting the Probes on the Microarray 8 X 4 Print Head microarray slide plate with wells holding probes in solution...
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- Spring '08