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DNA Array Analysis: Gene Expression Profiling Method

Learn how DNA arrays are used to assess gene expression levels in control and experimental cells. Step-by-step instruction from mRNA extraction to result interpretation with color-coded cDNA. Visit the link for an example array image.

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DNA Array Analysis: Gene Expression Profiling Method

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

  2. 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.

  3. cDNAgene 1 cDNAgene 2 cDNAgene 3 cDNAgene 4 cDNAgene 5 DNA Array: Single-stranded copy DNA foreach gene in a different well.

  4. 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.

  5. 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)

  6. 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.

  7. Control cDNA Experimental cDNA

  8. Methods: Step 5: Extract a smallamount in a pipette.

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

  10. Methods: Step 7: Extractmore cDNA and … … insert intosecond well, etc.

  11. Methods: Step 8ff: Continue until all wells arefilled.

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

  13. 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 (expressedin both experimental and control cells)

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

  15. Only in controlcells Mostly in controlcells Mostly in experimentalcells Only in experimentalcells Same inboth 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

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