Example of a DNA Array (note green, yellow red colors; also note that only part of the total array is depicted) http://www.biomed.miami.edu/arrays/images/agilent_array.jpg

Methods: Step 1: Create a DNA array (gene “chip”) by placing single-stranded DNA for each gene to be assayed into a separate “well” on the chip.

DNA Array: Single-stranded copy DNA for each gene in a different well. cDNA gene 1 cDNA gene 2 cDNA gene 3 cDNA gene 4 cDNA gene 5

Methods: Step 2: Extract mRNA from biological tissues subjected to an experimental treatment and from the same tissue subjected to a control treatment. Step 3: Make single-stranded DNA from the mRNA using “color coded” nucleotides.

Extract mRNA from Control Cells Extract mRNA from Experimental Cells Make single-stranded cDNA using green nucleotides Make single-stranded cDNA using red nucleotides cDNA = complementary DNA (DNA synthesized from RNA)

Methods: Step 4: After making many DNA copies of the RNA, extract an equal amount of cDNA from the controls & experimentals and place it into a separate container.

Control cDNA Experimental cDNA

Step 5: Extract a small amount in a pipette. Methods: Step 5: Extract a small amount in a pipette.

Step 6: Insert into first well. Methods: Step 6: Insert into first well.

… insert into second well, etc. Methods: Step 7: Extract more cDNA and … … insert into second well, etc.

Step 8ff: Continue until all wells are filled. Methods: Step 8ff: Continue until all wells are filled.

Step 9: Allow to hybridize, then wash away all single-stranded DNA. Methods: Step 9: Allow to hybridize, then wash away all single-stranded DNA.

Result: Some wells have no color-coded cDNA (no mRNA in either type of cell) Some wells have only red (i.e., expressed only in experimental cells) Some wells have only green (i.e., expressed only in control cells) Some wells have both red and green in various mixtures (expressed in both experimental and control cells)

Methods: Step 10: Scan with a laser set to detect the color & process results on computer.

Gene not expressed in control or in experimental cells Results: The colors denote the degree of expression in the experimental versus the control cells. Gene not expressed in control or in experimental cells Only in control cells Mostly in experimental cells Same in both cells