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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:

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.

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. # 162-0112) 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.

•  10 mg/ml phenolphthalein in Ethanol (RED)

•  10 mg/ml thymolphthalein in Ethanol (BLUE)

•  1:1 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. In the cancer slide we spotted with DNA that is known to be expressed in normal tissues and can be aberrantly expressed in cancer cells. The aberrant expression however does not have to be due to a mutation in that gene. Therefore more that 5 or 6 genes may be differentially expressed in the cancer tissue compared to the normal tissue.

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:

Footnotes

1 Genes that play a role in normal replication, transcription and translation may be upregulated in tumor cells that are dividing rapidly

2 Genes involved in normal aerobic respiration may be upregulated in tumor cells as their metabolism increases to meet energy requirements.

3 Cancer cells often upregulate genes involved in glycolysis as they outgrow the oxygen supply from surrounding blood vessels. Some of the most dangerous cancers display the so-called "glycolytic phenotype", marked by upregulated glycolysis even in the presence of ample oxygen for respiration.

4 Deficiencies in this tumor supressor gene are very common in Breast Cancer, for which the gene is named. It is the focus of much research.

5 Cytokines, growth factors/mitogens, and their receptors are often overexpressed in cancers. The signaling in which they are involved often leads to cell division.

6 Another common oncogene. Since its normal role is to suppress apoptosis, an overexpression may prevent cancer cells from destroying themselves.

7 This gene works in the same pathway as BCL2, except its role is to promote apoptosis. Deficiencies could help otherwise doomed cancer cells to persist.

8 The most common problems associated with p53 in cancer result from mutations, not deficiencies in gene expression. This patient may still very well have such a mutation, but that would not likely show up in a microarray analysis such as this, since the gene could still be expressed at normal levels.

9 This proto-oncogeneis often overexpressed in breast cancers, leading to invasion, metastasis and invasion.

10 This gene methylates DNA, thereby silencing transcription. An overexpression of this gene could lead to silencing of key tumor suppressor genes, like RASSF1.

11 MMP3 is thought to be activated by low pH. Degradation of the extracellular matrix aids cancer cells in invasion and metastasis. This is often seen in oxygen-deficient (hypoxic) cancers, where an increased rate of glycolysis leads to production of acid. It follows that MMPs are also commonly upregulated in cancers of the "glycolytic phenotype."

12 Rapidly growing tumors need to recruit more blood vessels to obtain nutritional requirements. It makes sense that this gene is activated by low O2 levels (hypoxia).

13 An upregulation of this gene would not be unusual in cancer, since this turns off a gene that turns off glycogen phosphorylase, which activates glycolysis.

14 Underexpression may be seen, as this gene would otherwise act to turn off growth signals.

15 Deficiencies in this gene could lead to diminished ability to turn off growth signals that originate at receptor tyrosine kinases (a common type of growth factor receptor).

16 Methylation of this gene's promoter region, possibly resulting in RASSF1 silencing, is seen in 95% of breast cancer cases. DNA Methylation is carried out by DNMT.

17 These genes all play a role in common signaling pathways that result in growth/proliferation. They are common proto-oncogenes that may be overexpressed in many cancers.

18 Cyclophosphamide is a common chemotherapy drug. Though expressed normally here, upregulation of this gene would make that drug a bad choice for the patient. Unfortunately, doctors rarely have this patient-specific information available to aid them in making decisions about therapeutic regimens.

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BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona September 18, 2000 Last Modified March 1, 2002 http://biotech.biology.arizona.edu