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The Big Idea

Present Genome Projects have accomplished sequencing entire genomes of many organisms.   What can we do with this information?   With all of the genes of an organism sequenced, a researcher can now look at the "expression" of all of the genes within a cell type or at a certain condition and compare that to another condition.    Most characteristics we see in an organism, most responses that cells mount due to a stimulation or cue, are not single gene phenomena.   Traditional genetics have used the "one gene, what is the one effect?" approach to trying to figure out what each gene does.   A new technique call Microarray, can allow us to look at up to 20,000 genes at one time and ask what all of these genes are doing in response to a particular stage of development or in reaction to an environmental stimuli.

Today's activity will be using microarray technology to look at the subset of an organism's genome (50 genes from a plant called Arabidopsis ) and will compare the expression of these genes during and in the absence of a common stress experienced by plants, heat.

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

10,000-1,000

1,000-100

<100

Of those genes, how many have known functions?  

How does one determine the function of a gene?  

Microarray Technology: 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 a Arabidopsis plant that has been grown under ideal Arabidopsis conditions and from a Arabidopsis plant that was stressed with hot temperature of 42°C for 4 hours.   All of the mRNA from each plant was reverse transcribed into DNA.   The mRNA from the normally grown Arabidopsis was transcribed into DNA with a blue dye label, whereas the mRNA from the heat stressed Arabidopsis was transcribed into DNA with a red label.   These DNAs can be mixed together and applied to the microarray slide with the 50 Arabidopsis genes.

Before beginning this activity be sure to watch an animation on microarrays using yeast grown under the conditions of with and without oxygen.  

http://occawlonline.pearsoned.com/bookbind/pubbooks/bc_mcampbell_genomics_1/medialib/method/chip/chip.html

Materials for the activity:

•  Microarray slide -- this slide contains a portion of the plant genome

•  Disposable pipet

•  cDNA mixture solution -- these cDNAs were made from Arabidopsis grown under normal conditions (attached to blue dye) and Arabidopsis grown under heat stress (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

1. Get a microarray slide, a disposable pipet, a tube labeled cDNA and a paper towel.
2. Place the slide onto the paper towel.
3. Add enough of the cDNA solution to the slide to completely cover it, but not spill off of the slide.
4. Let the cDNA hybridize with the microarray slide for 5 minutes.  
5. After the 5 minute incubation of the microarray slide with cDNA, rinse off the excess cDNA with the microarray wash solution (in squeeze bottle).
6. 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.
7. Record you data.

   Draw your results of the microarray:

Which genes were expressed during heat stress?   Which genes were expressed only during heat stress?

Color in the spots in the above pictorial representation of a slide with the appropriate corresponding color that you see on your slide.

If you were a researcher and found that a gene on your array, which has no known function, is expressed during heat stress and not otherwise, what could you do to determine the function of this gene.....

Which genes are not expressed during heat stress?   Why would some genes not be turned on during heat stress?   Can you think of any other conditions that you might see a difference in genes being expressed?    Please list or discuss.

Gene list

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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