DNA microarray flow chart and predictions Mrs. Stewart Medical Interventions Central Magnet School

 How does a DNA Microarray illustrate the differences in gene expression between two tissue samples?

 DNA Microarray Video DNA Microarray Video

 Create a flowchart for the steps in a DNA Microarray in your lab journals

Print single- stranded DNA gene sequences onto a microarray (slide) Isolate mRNA from normal and “experimental” cells Prepare flourescent (labeled) cDNA from each of the mRNA samples

Hybridize (place labeled cDNA sequences onto printed slides with known genes – complementary sequences will bind) Visualize results Analyze

 Grandpa Joe, Judy Smith’s father, has been a smoker for the past thirty years. Last year, Grandpa Joe came down with a cold that turned into pneumonia. It took him more than a month to recover. The family is very concerned he is going to develop lung cancer. They heard about a study being conducted at the local hospital that is exploring lung-cancer associated genes in smokers and non-smokers. The family convinces Grandpa Joe to participate in the study in order to learn more about his risk for developing lung cancer.

 The study is investigating six genes thought to be involved with lung cancer using DNA microarray technology. The researchers hope to compare gene expression of the six genes of interest between smokers and non-smokers in order to gain more knowledge of what causes a normal lung cell to become cancerous. You have been assigned to the study. Your first task is to learn more about the six genes of interest.

Gene 1: Human carcinoembryonic antigen (CEACAM6) This gene codes for a protein that is located in the extracellular matrix. This protein is involved with adhesion between cells and is thought to be a proto-oncogene and when over-expressed is an oncogene. Gene 2:Surfactant protein B (SFTPB) This gene codes for an extracellular protein. This protein enhances the rate of spreading and increases the stability of pulmonary surfactant, a lipid-rich material that prevents lung collapse by lowering surface tension at the air-liquid interface in the alveoli of the lungs.

Gene 3: P53 tumor suppressor (TP53) This gene codes for a protein that is located in the mitochondria and in the nucleolus. This protein is involved with cell cycle checkpoints. This gene is a tumor suppressor gene and is thought to be the “Guardian of the Genome.” Gene 4:SRYThis gene codes for a protein that is located in the nucleus. The protein that this gene codes for is testis- determining factor (TDF) which initiates male sex determination. This protein has no function in lung cells.

Gene 5: Cytochrome P450 (CYP1A1) This gene codes for a protein that is located in the endoplasmic reticulum. The protein catalyzes reactions involved in drug metabolism and synthesizes cholesterol, steroids, and other lipids. The expression of this protein is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke. Gene 6:Glypican 3(GPC3)This gene codes for a protein that is located in the plasma membrane and extracellular matrix. The gene controls cellular response to damage and may control cellular growth regulation and apoptosis. This gene is considered to be a tumor suppressor gene for lung cancer.

 Place one drop from each gene onto the correspondingly numbered spot on the microarray slide  These will harden in one minute  These spots represent the single-stranded DNA from each of the genes

 Obtain a cDNA dropper bottle (Hybridization Buffer) and place one drop onto each of the numbered spots on the microarray slide  Careful!! Do not touch the tip of the bottle to the DNA spots  The cDNA dropper bottle contains a solution of labeled cDNA from Grandpa Joe’s lung cells and a non-smoker’s lung cells mixed together

 Place your DNA microarray slide onto a white piece of paper to observe results.  After you complete your student response sheet: Wipe off the six spots on your slide with a paper towel. Wash and dry your slide.

 Draw a diagram of the slide in your laboratory journal. Make sure to clearly indicate which gene is on which spot.  Draw your results on the Student Response Sheet. Include a description of the color of each spot. Analyze the results.

Human carcinoembryonic antigen (CEACAM6) This gene codes for a protein that is located in the extracellular matrix. This protein is involved with adhesion between cells and is thought to be a proto-oncogene and when over-expressed is an oncogene. Surfactant protein B (SFTPB) This gene codes for an extracellular protein. This protein enhances the rate of spreading and increases the stability of pulmonary surfactant, a lipid-rich material that prevents lung collapse by lowering surface tension at the air-liquid interface in the alveoli of the lungs. P53 tumor suppressor (TP53) This gene codes for a protein that is located in the mitochondria and in the nucleolus. This protein is involved with cell cycle checkpoints. This gene is a tumor suppressor gene and is thought to be the “Guardian of the Genome.” SRY This gene codes for a protein that is located in the nucleus. The protein that this gene codes for is testis-determining factor (TDF) which initiates male sex determination. This protein has no function in lung cells. Cytochrome P450 (CYP1A1) This gene codes for a protein that is located in the endoplasmic reticulum. The protein catalyzes reactions involved in drug metabolism and synthesizes cholesterol, steroids, and other lipids. The expression of this protein is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke. Glypican 3(GPC3) This gene codes for a protein that is located in the plasma membrane and extracellular matrix. The gene controls cellular response to damage and may control cellular growth regulation and apoptosis. This gene is considered to be a tumor suppressor gene for lung cancer.

 Pink result = Gene transcription (expression) is induced  Blue result = Gene transcription (expression) is suppressed  Purple result = Gene transcription (expression) is not affected  Clear/white result = Gene not expressed **These colors differ from a regular DNA microarray in which Red, Yellow and Green are used.

 When the ratio is greater than one, the gene is induced by tumor formation. This means that the gene transcription was more active in cancer cells than in normal cells.  When the ratio is less than one, the gene is suppressed by tumor formation. This means that the gene transcription was less active in cancer cells than in normal cells.  When the ratio is equal to one, the gene is not affected by tumor formation. This means that the gene transcription was the same in cancer cells as it was in normal cells.  When the ratio is zero, the gene is not expressed in either cell.

 How does a DNA Microarray illustrate the differences in gene expression between two tissue samples?