Download Get a microarray slide, a disposable pipet, a tube

The Big Idea
Over the last 28 years many defects in genes have been linked to cancer, each promising to be the
magic in understanding and curing cancer. The understanding now indicates cancer as a
multistep process, each of these steps generally due to a genetic aberration. Accumulation of
these mutations in genes allows the cell to progress to tumor and malignancy.
Every cancer can be attributed to a different set of genetic aberrations, and different genes are
either expressed or not expressed. More than 100 different types of cancer can be found within
specific organs. Each caner has a different potential of being treated by different therapies. For
example, it has been shown cancer cells that lack the p53 protein do not respond well to radiation
therapy, and other non-malignant cells lacking p53 will readily progress to malignancy in
response to radiation. Thus the treatment itself causes more cancers.
The best way to treat a cancer then would be to know which genes are mutated and which genes
are expressed or not expressed in the tissue. One approach that would allow you to look at
numerous genes expressed and use that knowledge to determine treatment. Gene expression of
numerous genes can be looked at by a new technique called microarray analysis.
A central feature of today's molecular view of cancer is that cancer does not develop all at once,
but across time, as a long and complex succession of genetic changes. Each change enables
precancerous cells to acquire some of the traits that together create the malignant growth of
cancer cells.
Two categories of genes play major roles in triggering cancer. In their normal forms, these genes
control the cell cycle, the sequence of events by which cells enlarge and divide. One category of
genes, called proto-oncogenes, encourages cell division. The other category, called tumorsuppressor genes, inhibits it. Together, proto-oncogenes and tumor-suppressor genes coordinate
the regulated growth that normally ensures that each tissue and organ in the body maintains a size
and structure that meets the body's needs.
What happens when proto-oncogenes or tumor-suppressor genes are mutated? Mutated protooncogenes become oncogenes, genes that stimulate excessive division. And mutations in tumorsuppressor genes inactivate these genes, eliminating the critical inhibition of cell division that
normally prevents excessive growth. Collectively, mutations in these two categories of genes
account for much of the uncontrolled cell division that occurs in human cancers.
Before beginning this activity: watch an animation on microarrays.
http://learn.genetics.utah.edu/content/labs/microarray/
Be sure you clearly understand how microarrays are used to measure gene expression in certain
types of cells or tissues
Microarray Technology: Background for this activity
Microarray technology can place all of the genes that have been sequenced for an organism and
by simple hybridization ask which of the genes are producing mRNA (and therefore expressed) in
specific cells or tissues. The DNA of the sequenced genes is "spotted" onto a microscope slide.
Up to 20,000 genes can be analyzed at one time, but for this activity only 50 genes have been
spotted for your analysis. Messenger RNA was extracted from breast tissue that was biopsied
from a cancer. This will be compared to breast tissue that did not contain the caner. All of the
mRNA from each of the tissues was reverse transcribed into cDNA. The mRNA from the
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normally breast tissue was transcribed into DNA with a blue dye label, whereas the mRNA
from the breast cancer tissue was transcribed into DNA with a red label. These cDNAs can be
mixed together and applied to the microarray slide that has been adhered with genes of cellular
processes often found aberrant in breast cancers.
Materials for the activity:
 Microarray slide -- this slide contains genes involved in cancer
 Disposable pipette
 cDNA mixture solution -- these cDNAs were made from normal breast tissue (attached to
blue dye) and breast cancer tissues (attached to red dye).
 Wash solution
 Color developing reagent
You will look develop the microarray and then analyze which genes were "expressed" under
which conditions.
Procedure:
Get a microarray slide, a disposable pipet, a tube labeled cDNA and a paper towel.
1. Place the slide onto the paper towel.
2. Add enough of the cDNA solution to the slide to completely cover it, but not spill off of
the slide.
3. Let the cDNA hybridize with the microarray slide for 5 minutes.
4. After the 5 minute incubation of the microarray slide with cDNA, rinse off the excess
cDNA with the microarray wash solution (in squeeze bottle).
5. Add color solution, again enough to cover the slide but not spill over the slide. This
solution is toxic so take care to not get it on you, and wash off of skin immediately. Let
the color solution set for 30 sec, then wash off excess with microarray wash solution.
6. Record you data.
Draw your results of the microarray:
Color in the spots in the above pictorial representation of a slide with the appropriate
corresponding color that you see on your slide
Which genes were expressed only in cancer tissue (red)
Which genes are expressed only in healthy tissue cell(blue)? Why would some genes not be
turned on in cancer tissue?
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Go to http://www.ncbi.nlm.nih.gov/
Search the “Gene” data base for the genes that were expressed in the cancer cells, but not in the
healthy cells. Ie: GLUT1 Use the symbol for the gene to search the “gene” database
List the chromosome that this gene is found on- in humans and list the general function of the
gene.
Repeat for genes expressed in the healthy tissue, but not the cancer tissue
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Symbol
POL1
GAPdH
HK1
ALDH1
GLUT1
ACTG1
DNASE1
RNASE4
TOP1
BRCA1
PDGFR
CYP1A1
BCL2
LIG1
POL1
APAF1
p53
ZNF84
MUC1
G6PD
TNF
ADH4
DNMT1
POLR2A
MDM2
MMP3
VEGF
ACAT1
MCR4
PDK2
GPB
DUSP1
PRL1
JUN
FOS
RASSF1
Name
DNA Polymerase
Glyc.Ald.Phos.DeHHexokinase 1
AldDeHase
Glucose Transporter 1
Actin, cytoplasmic
Deoxyribonuclease I
Ribonuclease 4
Topoisomerase I
Breast cancer type 1 susceptibility protein
Platelet-derived Growth Factor Receptor
Cytochrome P450 1A1
B-cell lymphoma protein 2
DNA Ligase I
DNA Polymerase Iota
Apoptosis Protease Activating Factor 1
p53 (tumor protein 53)
Zinc Finger Protein 84
Transmembrane Mucin 1
Glu.6-phosp DeH-ase
Tumor necrosis factor
Alcohol Dehydrogenase
DNA Methyltranferase I
RNA Polymerase, subunit 2
MDM2
Matrix Metalloprotease 3 (Stromelysin)
Vascular endothelial growth factor
Acetoacetyl-CoA thiolase
Melanocortin receptor
Pyruvate DeH-ase Kinase
Glycerol Phosphatase Beta
Dual-specificity protein 1
Protein Tyrosine Phosphatase
Jun
Fos
Ras-association domain, family 1 protein
Function
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43
44
45
46
47
48
49
50
RAS
SOS
EGFR
CS
AChE
CDKNA
IL6
GSTP1
VEGFR
PLCG1
MYC
RPS18
NAT1
MAPK
ras
sos
Epithelial Growth Factor Receptor
Citrate Synthase
Acetylcholinesterase
p21
Interleukin 6
Glutathione S-transferase
Vascular Endothelial Growth Factor Rec.
Phospholipase-C gamma
c-Myc Proto-oncogene
Ribosome Subunit 18S
n-acetyltransferase
Mitogen-activated Protein Kinase
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