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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:

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.  

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.

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 Arabidopsis slide:

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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