文档介绍：Microarrays for Gene Expression Analysis Questions: What genes are expressed in this tissue under these conditions? What genes are expressed in my treated cells versus the control? What genes are expressed during the phases of the cell cycle? What genes are expressed in diseased tissue versus normal tissue? Microarrays – other uses Questions: What point mutations exist and what bases are located at the substitution positions? What bases are substituted where there are multiple mutations very close together? Which allele of this gene do we have? Is this the mutant or wildtype? Goals Finding Co-Regulated Genes Understanding Gene works Expressed Genes = mRNA DNA messenger RNA protein Expressed Genes = Currently Transcribed mRNA Extract RNA mRNA mRNA mRNA mRNA Isolate mRNA’s Affymetrix Oriented Fluorescently tagged cRNA One chip per sample One for control One for each experiment Other methods include two dyes/one chip Red dye Green dye Control and experiment on same chip Creating Targets mRNA Reverse Transcriptase cDNA PCR Amplification of DNA In Vitro transcription to create cRNA RNA-DNA Hybridization probe sets DNA (25 base oligonucleotides of known sequence) Targets RNA Non-Hybridized Targets are Washed Away “probe sets”(oligo’s) Targets (fluorescently tagged) Non-bound ones are washed away Picture of Gene Chip