So let's look at how this would actually work in a real experiment. mRNA is isolated from yeast cells in state A(e.g, minus galactose) and from yeast cells in state B(e.g, plus galactose), and copies of each population is made such that one fluoresces red and the other fluoresces green. After mixing, these fluorescent molecules are hy bridized to the slides containing N5, 800 DNa probes, each one specific for detecting hybridization of many copies of an individual mRNA species yeast in state A yeast in state B What's happening at each spot? ↓↓ 88 T oo。ooo MITT Hybridization The location and identity of each probe on the microarray slide is known, and each probe is specific for a single mRNA. the color and intensity of the fluorescence is measured by scanning the slide with lasers, and the relative abundance of each mrna in the cells of state A vs State b can be calculated from the RNA present much higher in State A than state B emitted fluorescence, i.e the relative level of mRNA present much higher in State B than State A 5,800 mRNAs can be compared between two mRNA present at equal levels in States A and B populations of yeast cells Presenting data for thousands of mrna transcripts is clearly a challenge. You could present endless tables of data, but our brains are much more adept at recognizing shapes, patterns and colors. Colored representations of up and down regulation of transcripts levels is the preferred way to present data Northern Blot vs. Microarray Each colored vertical line in the horizontal lane displays the relative expression level of a single mRNA Images removed due to copyright reasons. Please see Lodish, Harvey, et al. Molecular Cell Biology. 5th ed. New York: W.H. Freeman and Company, 2004So let’s look at how this would actually work in a real experiment. mRNA is isolated from yeast cells in state A (e.g., minus galactose) and from yeast cells in state B (e.g., plus galactose), and copies of each population is made such that one fluoresces red and the other fluoresces green. After mixing, these fluorescent molecules are hybridized to the slides containing ~5,800 DNA probes, each one specific for detecting hybridization of many copies of an individual mRNA species. Yeast in state A AAAAA AAAAA AAAAA AAAAA TTTTT TTTTT TTTTT TTTTT Yeast in state B AAAAA AAAAA AAAAA AAAAA TTTTT TTTTT TTTTT TTTTT Label copies of mRNA species with RED or GREEN Isolate mRNA populations MIX TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT Hybridize to the microarray Yeast in state A AAAAA AAAAA AAAAA AAAAA TTTTT TTTTT TTTTT TTTTT Yeast in state A AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT Yeast in state B AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT Label copies of mRNA species with RED or GREEN Isolate mRNA populations MIX TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTTTTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT Hybridize to the microarray What’s happening at each spot? Hybridization What’s happening at each spot? Hybridization The location and identity of each probe on the microarray slide is known, and each probe is specific for a single mRNA. The color and intensity of the fluorescence is measured by scanning the slide with lasers, and the relative abundance of each mRNA in the cells of State A vs State B can be calculated from the emitted fluorescence. i.e., the relative level of 5,800 mRNAs can be compared between two populations of yeast cells. mRNA present much higher in State A than State B mRNA present much higher in State B than State A mRNA present at equal levels in States A and B mRNA present much higher in State A than State B mRNA present much higher in State B than State A mRNA present at equal levels in States A and B Presenting data for thousands of mRNA transcripts is clearly a challenge. You could present endless tables of data, but our brains are much more adept at recognizing shapes, patterns and colors. Colored representations of up and down regulation of transcripts levels is the preferred way to present data. Northern Blot vs. Microarray Each colored vertical line in the horizontal lane displays the relative expression level of a single mRNA Each colored vertical line in the horizontal lane displays the relative expression level of a single mRNA Images removed due to copyright reasons. Please see Lodish, Harvey, et. al. Molecular Cell Biology. 5th ed. New York : W.H. Freeman and Company, 2004