Activity 3.1.4 DNA Microarray
Introduction
We have approximately 20,000 to 25,000 genes. Traditionally, molecular biologists could
only study one gene at a time. Using this approach to investigate each individual gene in
the human genome would have taken a very long time. Advances in technology now allow
scientists to look at thousands of genes all at once using a tool called a DNA microarray
(also called a DNA chip or gene chip). Remember that every cell in our body (with a few
exceptions such as red blood cells) have copies of each of our 20,000 genes. If all of the
body’s cells have the exact same DNA, then what makes a skin cell different from a nerve
cell? The difference is in what genes are turned “on” and “off” in each cell. In a skin cell,
the genes for producing melanin, a protein that gives your skin color, are turned on. In a
nerve cell, the genes for producing the neurotransmitter acetylcholine are turned on. The
genes for producing the hormone insulin are turned off in both skin and nerve cells.
DNA microarrays work by measuring the amount of mRNA for every gene that is present
in a cell sample so that scientists can determine which genes are turned on and which are
turned off. The reason scientists measure the amount of mRNA for every gene is the
basic assumption that if a gene is being transcribed to mRNA, it is considered to be
expressed. Scientists can then compare these results to those found in another sample to
determine the differences in gene expression between the two samples.
How are DNA microarrays made? Using the information learned from the Human Genome
Project, scientists design primer pairs so that they can use PCR to make copies of every
gene in the human genome. They then separate the double-stranded DNA from each
gene copy into single strands and place microscopic droplets of each single-stranded
DNA sample into ordered rows and columns on a glass, plastic or silicon slide. This is
called the DNA microarray. Each DNA microarray can contain tens of thousands of genes.
Let’s take a look at cancer cells. Remember that cancer involves mutations with the genes
that regulate cell growth, division, and death. To be able to better diagnose, understand,
and treat cancer, it is important to understand what goes wrong with the gene expression
in cancer cells. DNA microarray technology allows scientists to use the mRNA taken from
two cells to determine which genes are turned on and which genes are turned off.
Scientists can therefore use DNA microarray technology to help us learn about the
differences in gene expression between a healthy cell and a cancer cell. In a DNA
microarray experiment, the two cell samples can either be taken from the same patient or
be taken from different patients, depending on the purpose of the study.
In this activity, you will explore DNA microarray technology. As you learn about the
science behind this technology, you will perform a simulated DNA microarray to study the
gene expression in a smoker’s versus a non-smoker’s lung cells and analyze the DNA
microarray results.
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 1
Equipment
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Computer with Internet access
 Micropipettor (0.5-10 µl)
Activity 3.1.4: Student Response
 Disposable micropipette tips
Sheet
 70°C hot water bath
Laboratory journal
 Digital camera (optional)
Permanent marker
 Safety goggles
Colored pencils or markers
 Latex or nitrile exam gloves
Micropipettor (20-200 µl)
Carolina Biological DNA CHIPS: Genes to Disease Kit
o Glass slide
o Genes 1-6 Dropper Bottles
o cDNA Mixture Dropper Bottle
Procedure
Part 1: DNA Microarray Virtual Lab
1. Obtain a Student Response Sheet from your teacher.
2. Go to the “DNA Microarray Virtual Lab” found at the University of Utah’s Learn.Genetics:
Genetic Science Learning Center’s website:
http://learn.genetics.utah.edu/content/labs/microarray/
3. Click on the picture of the microarray to begin.
4. Click on “Chapter 3: The Experiment!” to begin the virtual experiment.
5. Answer the questions on your Student Response Sheet as you work through the virtual
DNA microarray experiment.
6. Answer Conclusion questions 1 – 3.
Part II: Smoking and Lung Cancer
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.
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 2
Below are descriptions for the six genes of interest:
Gene Name
Protein Function:
(and Symbol):
This gene codes for a protein that is
Human
located in the extracellular matrix.
Gene carcinoembryoni This protein is involved with adhesion
1:
c antigen
between cells and is thought to be a
(CEACAM6)
proto-oncogene and when overexpressed is an oncogene.
This gene codes for an extracellular
protein. This protein enhances the
rate of spreading and increases the
Surfactant
Gene
stability of pulmonary surfactant, a
protein B
2:
lipid-rich material that prevents lung
(SFTPB)
collapse by lowering surface tension
at the air-liquid interface in the alveoli
of the lungs.
This gene codes for a protein that is
located in the mitochondria and in the
P53 tumor
nucleolus. This protein is involved
Gene
suppressor
with cell cycle checkpoints. This gene
3:
(TP53)
is a tumor suppressor gene and is
thought to be the “Guardian of the
Genome.”
This gene codes for a protein that is
located in the nucleus. The protein
Gene
that this gene codes for is testisSRY
4:
determining factor (TDF) which
initiates male sex determination. This
protein has no function in lung cells.
This gene codes for a protein that is
located in the endoplasmic reticulum
and catalyzes reactions involved in
drug metabolism and synthesizes
Gene
Cytochrome
cholesterol, steroids, and other lipids.
5:
P450 (CYP1A1)
The expression of this protein is
induced by some polycyclic aromatic
hydrocarbons (PAHs), some of which
are found in cigarette smoke.
This gene codes for a protein that is
located in the plasma membrane and
extracellular matrix. The gene
Gene
Glypican
controls cellular response to damage
6:
3(GPC3)
and may control cellular growth
regulation and apoptosis. This gene
is considered to be a tumor
suppressor gene for lung cancer.
Prediction:
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 3
7. Highlight or underline any interesting or important information about the function of each
protein.
8. Predict how these genes will be expressed in a DNA microarray of a smoker versus a
non-smoker. Would you expect the genes to be induced in the smoker (more expressed),
suppressed in the smoker (less expressed), not expressed in either the smoker or the
non-smoker, or expressed the same in both the smoker and non-smoker?
9. Record your predictions in the Prediction column of the above table.
Part III: Microarray Wet Lab
Now that you know more about the six genes of interest, your job is to perform a simulated
DNA microarray using tissue samples taken from Grandpa Joe and a non-smoker’s tissue
samples. The cDNA has already been prepared for you. You will first prepare the simulated
DNA microarray by spotting each of the six gene sequences onto a glass slide. Actual DNA
microarrays have thousands of microscopic DNA spots on the slide. In this lab, our spots will
be much larger than in a regular microarray, and you will be able to view the spots without
specialized equipment.
10. Put on safety gloves and goggles.
11. Use the permanent marker to number the six clear spots on the slide Genes 1-6. Make
sure not to touch the surface of your slide (handle it only by the edges).
12. Load 30 µl of Gene 1 onto the corresponding spot on your slide. You will need to remove
the top off the dropper bottle. Do this for each of the 6 genes. Use a fresh tip for each
gene. Your spots will harden in less than one minute.
o These spots represent the DNA sequences from six different genes.
13. Draw a diagram of the slide in your laboratory journal. Make sure to clearly indicate which
gene is on which spot.
14. Obtain a cDNA dropper bottle (Hybridization Buffer) from your teacher and carefully add
10 µl to each spot on your slide. Do not allow the micropipette tip to touch 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. You cannot see the color
because the cDNA is very dilute. When added to the printed microarray slide, the
labeled cDNA in the solution will pair up with the complementary DNA for each gene
spotted onto the microarray, according to the base pair rules. As each cDNA binds to
the appropriate DNA spot on the slide, the labeled cDNA becomes concentrated in
that spot, allowing the spot to be visualized.
 The cDNA used in an actual microarray is labeled with red and green fluorescent
dyes and the colors must be viewed using a fluorescent scanner to measure the
intensity of each spot. In this lab, the cDNA is labeled with pink (Grandpa Joe’s lung
cells) and blue (non-smoker’s lung cells) and do not need a fluorescent scanner to
view the results.
15. Place your DNA microarray slide onto a white piece of paper to observe results.
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 4
16. Draw your results under question 11 on the Student Response Sheet. You should also
take a photo of your slide. Include a description of the color of each spot.
17. Answer questions 12 – 16 on the Student Response Sheet.
18. Wipe off the six spots on your slide with a paper towel. Wash and dry your slide.
You have just characterized the expression level of each gene from a smoker’s and nonsmoker’s tissue subjectively (i.e. deep blue versus light blue, deep pink versus light pink, etc).
When scientists analyze microarrays, they need to be able to quantitatively measure the
results. First scientists convert the colors to numbers according to the intensity of red and
green. For example, look at the following DNA microarray results for four genes:
Gene A:
Gene B:
Gene C:
Gene D:
Red (Tumor
Cells)
Green (Normal
Cells)
Superimposed
Image of Green
and Red
Now we will convert these colors into numbers. The numbers represent the intensity of the
red or green color. The brighter the color, the larger the number to represent it. For example,
a value of 400 indicates a very bright light intensity, whereas, a value of 100 is a dull color:
Red
(Tumor Cells)
Green
(Normal Cells)
Gene A:
Gene B:
Gene C:
Gene D:
400
200
100
200
100
300
100
400
The next step is to calculate the ratio of red to green for each gene:
Gene A:
Gene B:
Gene C:
Ratio
400:100 =
200:300 =
100:100 =
Red: Green
4:1 = 4
2:3 = 0.67
1:1 = 1
(Tumor:Normal)
Gene D:
200:400 =
2:4 = 0.5
The ratios can be used to give meaning to the results:
 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.
19. Answer Conclusion question 4.
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 5
Now you will do this same process with the data you collected in your microarray. The scale
below represents the different shades you might see in your microarray. The shades range
from blue to pink as you go from left to right on the scale. Remember that in our microarray,
the cDNA from Grandpa Joe’s lung cells were labeled pink and the cDNA from the nonsmoker’s lung cells were labeled blue. The numbers above the scale represent the gene
expression ratios that correspond with each color.
20. Match the colors in your microarray to those in the above scale. Your colors may not be of
the same intensity as those shown above. The colors you see will not match exactly with
the chart. Estimate the ratios as best you can, selecting ratios between the given numbers
whenever necessary.
21. Record your gene expression ratios for your microarray data under number 17 on your
Student Response Sheet.
22. Compare your results to your predictions. Were your predictions for each gene correct?
Explain your findings in your laboratory journal.
23. Answer the remaining Conclusion questions.
Conclusion Questions:
1. Imagine you want to learn more about Mike Smith’s osteosarcoma. How could you use
microarray technology to determine which genes have been affected in his tumor cells?
2. What does it tell us if two genes show the same levels of expression in cancer cells and
normal cells?
3. What does it tell us if there are some genes that are highly expressed in normal cells but
not expressed in cancer cells?
4. What range of ratios could indicate that a gene was not expressed in cancerous tissue,
but was expressed in healthy tissue?
5. When analyzing DNA microarray results, why are colors turned into ratios?
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 6
6. When analyzing DNA microarray results, what does a lack of color indicate? (Note: in this
experiment you saw a lack of color as white, but in real microarrays it would be seen as
black.)
7. What further questions do you have about the gene expression differences between
Grandpa Joe’s tissue sample and the non-smoker’s tissue sample? How might you design
an experiment to answer one of your questions?
8. What can a DNA microarray teach us about oncogenes and tumor suppressor genes?
9. Use the information learned from the DNA microarray to write an argument to convince
Grandpa Joe to stop smoking.
Web Portfolio:
1) Give a short but complete definition of what a microarray is AND what it does.
2) Include a picture of your microarray slide and a well written description of the color of
each spot and what the color indicates.
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 7