Microarray activity: Teacher’s Guide
This Microarray is a simulation using pH indicators phenolphthalein and thymolphthalein.
These pH indicators will change from clear to either red or blue, respectively at pH>8. Normal
glass slides are used, nitrocellulose, acting as the solid support for the indicators (DNA), is glued
to the slide. Nitrocellulose, which is positively charged, will bind negatively charged molecules.
The negatively charged indicators are “spotted” onto the nitrocellulose. True Microarrays utilize
fluorescent dyes on the cDNA to differentiate the two different conditions of the analysis. Gene
expression can be assessed based on the intensity of the green or red fluorescence. The
simulation dye method only allows us to measure a gene as on in one condition (red) or the other
condition (blue) or on in both conditions (purple).
Some questions to think about before the activity:
Approximately how many genes make up a typical multicellular organism?
>1 million
1 million-100,000
100,000--10,000—for most eukaryotes this is the correct answer
10,000-1,000
1,000-100
<100
Humans have ~25-30,000 genes, which is what is also found in mouse and in Arabidopsis.
Fruit fly has ~13,000 genes. Yeast (also a eukaryote) has 6,000 genes. E.coli has 3,200
genes.
Of those genes, how many have known functions?
Probably only 1,000-2,000 genes have known function in these well studied organisms.
How does one determine the function of a gene?
Genes are portions of chromosomal DNA that contain the message and other information
necessary to produce a protein. The protein, once present in the cell, carries out a function
unique to the cell. A researcher can look for the function of a gene in two distinct ways: 1)
what does the protein do in the cell. For example if the protein spans the lipid membrane
and has an electrochemical function of transporting ions across the membrane, then the
gene may be an ion channel or transporter. And, 2) what is the physical external effect
(phenotype) of loosing that gene on the organism. For example, the gene to produce hair
from the follicles produces a protein that if missing will result in a completely hairless
person, external physical effect is hairlessness, therefore the gene is call hairless. But the
role the protein carries out in the cell may not be a direct link to the protein that you see on
the top of your head. The protein, hairless, regulates other genes in the cell one of which is
the hair protein. Our hairless gene may tell the cell when and where (and which cells) to
have other proteins produced. Addressing hairless function, for 1) what does the protein do
in the cell--it regulates to the production of other proteins, including hair protein.
Addressing the characteristic of an individual with a defective hairless gene, the external
effect would be the complete absence of hair. The study of human genetics has been
investigated primarily with the discovery of diseases, but to remove a gene function and
induce a disease in a human could not (should not) be done. One can however do this with
plants, for example, genetically modify a plant and produce a plant that make many
underdeveloped flowers, you have made broccoli, or something reminiscent of broccoli.
Whereas the ethical implication of producing a mouse, or even a fruit fly with many
underdeveloped heads is greater.
How is cDNA made and used in microarray technology?
cDNA is a synthesized copy of messenger RNA, mRNA. Most eukaryotic mRNA has a
poly (A) tail, which can be used to purify the mRNA from the bulk of the cellular RNA.
Cellular RNA can be passed over a column to which deoxythymidine (dT) residues have
been attached. Poly (A) tail hybridizes to the oligo(dT), thus mRNA sticks to the column,
the rest of the cellular RNA runs through and mRNA is purified from the rest of the RNA.
mRNA can be converted to cDNA using reverse transcriptase (RT) and a polyT primer.
The RNA is degraded leaving single stranded cDNA left to hybridize with DNA stands
representing genes on the microarray slide. If cDNA binds to the DNA on the slide, then
that gene was expressed (the mRNA was present) in the tissue. Since we have placed
specific DNA on the slide, we can infer which genes are expressed.
When the cDNA is synthesized a fluorescent tag can be incorporated into the molecule that
can be easily detected, cDNA from one condition is tagged with red, and cDNA from
another condition is tagged green. Our simulation uses red and blue tags.
Which genetic aberrations have been implicated in cancer? What cellular functions are affected
(turned on or off) in cancer cells, and how might these affect normal cell development?
Many different genes have been found to linked to cancer, but it is not so much which
mutant gene, but which collection of mutated genes and in what order did these mutations
occur. Numerous genes could be involved, generally 5 or 6 genes are required for the cell(s)
to be considered malignant cancer. These genes fall into a specific set of capabilities. These
are characterized in a detailed review article that was published in 2000 'Hallmarks of
Cancer' by Hanahan and Weinberg. Scientific American's 'Untangling the Roots of
Cancer' provides another review on the subject.
The Microarray slide is a normal glass slide, with a piece of nitrocellulose (BioRad Cat. # 1620112) cut to fit the slide and glued with Elmer’s school glue onto the slide. Rapidograph pens
are utilized to “spot” the DNA (see solutions below) onto the nitrocellulose. I used a 0.8 mm
size, but a 1 mm should work well too. Rapidographs are used for art and can be purchased in
most art supply stores. Instead of filling the pen with ink we used the following solutions.
1) 10 mg/ml phenolphthalein in Ethanol (RED)
2) 10 mg/ml thymolphthalein in Ethanol (BLUE)
3) 2:1 mix of 10 mg/ml phenolphthalein in Ethanol and 10 mg/ml thymolphthalein in
Ethanol (PURPLE)
The slide can also be not spotted representing a gene that is not expressed in either of the types
of cells used in the experiment.
The slide is incubated with cDNA (this solution is water—make sure the water does not have a
pH of 8 or less.
The cDNA is washed off with microarray solution (again this is water—if someone asks, the
wash solution is normally a low salt and mild detergent to dissociate any weak interaction that is
not due to strong hybridization).
Color solution is 5% NaOH. Basic solution will react with the indicators to result in a color
change from clear to red, blue and purple. Take care with this solution, it is a strong base.
Wash solution is again water.
Results for slide:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Symbol
POL1 1
GAPdH 2
HK1 2
AT3g51970
At1g68600
AT5g43760
AT1G56600
AT5g20230
AT3g13772
At2g35650
At2g30020
At1g23480
AT4G27670
AT3g52940
At1g01580
AT4g25980
At1g80840
Name
DNA Polymerase
Glyc.Ald.Phos.DeH-ase
Hexokinase 1
wax synthase-like protein
expressed protein (not expressed in either condition)
beta-ketoacyl-CoA synthase
Galactinol synthase+++
blue copper binding protein
multispanning membrane protein
putative glucosyltransferase
protein phosphatase 2C, putative / PP2C, putative--hypothetical protein
HSP21+++
nuclear envelope membrane protein - like
hypothetical protein
putative peroxidase
WRKY family transcription factor---
Function
DNA replication
Krebs Cycle
Glycolysis
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
At2g01680
At1g74190
At1g74370
AT3g23150
At2g40940
At4g12400
At1g17240
At2g14860
AT4g33900
At2g02780
AT5g63410
At2g25250
AT5g14210
At2g34180
AT3g16300
At1g79780
At1g07710
At1g78720
At2g02590
At3g10930
AT5g07570
AT4g30430
AT3g12090
At1g54010
42
43
44
45
46
47
48
49
50
AT4g16590
AT5G07330
At1g04310
At1g17250
At1g49380
At1g44414
AT3g49050
At1g10340
At1g16680
#3 APX-1b
#7 Galactinol
synthase
#13 HSP21
#23 stress
inducible
protein-putative
#11 PP2C
putative
unknown protein
disease resistance protein
putative RING zinc finger protein
ethylene receptor
ethylene response sensor
stress-inducible protein, putative+++
putative receptor protein kinase
22 kDa peroxisomal membrane protein
putative protein
putative receptor-like protein kinase
putative protein
expressed protein--receptor protein kinase-like protein
putative protein kinase
hypothetical protein
hypothetical protein
hypothetical protein (not expressed in either condition)
protein transport protein sec61 alpha subunit, putative
putative transport protein
hypothetical protein--glycine/proline-rich protein
senescence-associated protein homolog
senescence-assocated protein
cytochrome P450, putative contains Pfam profile:
PF00067 cytochrome P450--cellulose synthase like protein
expressed protein+++
putative ethylene receptor
putative receptor protein kinase
hypothetical protein (not expressed in either condition)
hypothetical protein+++
calmodulin-binding heat-shock - like protein
hypothetical protein (not expressed in either condition)
hypothetical protein
aspirtate peroxidase- involved in electron transport,
responds to oxidative stress generally in the presence of
H2O2
involved in production of secondary metabolite, many
secondary metabolites are used in response to stress,
pathogen or predatory attack, or general well being of
plant
Small heat shock protein, transported to chloroplast,
expression in response to heat
Has similarity to another protein that is stress inducible,
no characterization, nothing
Known about its response to stress
Dephosphorylates proteins, used in signal transduction
on/off switch, in response to secondary metabolite, or
#17 WRKY
#41
cytochrome
P450
#39 and 40
senescence
protein
signal outside of cell, i.e. stress
Family of transcription factors (72 in Arabidopsis) have
some role in the regulation of plant specific
physiological programs, i.e. trichome development,
pathogen defense
Cytochrome P450 enzymes in biosyntheses of some
plant secondary metabolites defense compounds,
hormones and growth regulators, metabolitesherbicides, insecticides.
Senescence is the process of aging, in plants
chlorophyll declines, water weight reduced,
Nutrients are remobilized to other parts of the plant, i.e.
leaf senescence, nutrients relocated to flower or fruit-senescence can mimic with stress responses