Transcript DNA Microarray - Montana State University

DNA MICROARRAY
Jamie Mashek
What we will be discussing…
 What is DNA microarray?
 The purpose of using DNA microarray.
 The plate.
 Steps to perform a microarray.
 Benefits.
 Problems.
What is DNA Microarray?
 Scientists used to be able to perform genetic analyses of a few
genes at once. DNA microarray allows us to analyze thousands of
genes in one experiment!
Purposes.
 So why do we use DNA microarray?
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To measure changes in gene expression levels – two samples’ gene expression
can be compared from different samples, such as from cells of different stages of
mitosis.
To observe genomic gains and losses. Microarray Comparative Genomic
Hybridization (CGH)
To observe mutations in DNA.
The Plate.
 Usually made commercially.
 Made of glass, silicon, or nylon.
 Each plate contains thousands of spots, and each spot contains a
probe for a different gene.
 A probe can be a cDNA fragment or a synthetic oligonucleotide,
such as BAC (bacterial artificial chromosome set).
 Probes can either be attached by robotic means, where a needle
applies the cDNA to the plate, or by a method similar to making
silicon chips for computers. The latter is called a Gene Chip.
Let’s perform a microarray!
1)
Collect Samples.
2)
Isolate mRNA.
3)
Create Labelled DNA.
4)
Hybridization.
5)
Microarray Scanner.
6)
Analyze Data.
STEP 1: Collect Samples.
 This can be from a variety of organisms. We’ll use two
samples – cancerous human skin tissue & healthy human
skin tissue
STEP 2: Isolate mRNA.
 Extract the RNA from the samples. Using either a column, or a
solvent such as phenol-chloroform.
 After isolating the RNA, we need to isolate the mRNA from the
rRNA and tRNA. mRNA has a poly-A tail, so we can use a column
containing beads with poly-T tails to bind the mRNA.
 Rinse with buffer to release the mRNA from the beads. The buffer
disrupts the pH, disrupting the hybrid bonds.
STEP 3: Create Labelled DNA.

Add a labelling mix to the RNA.
The labelling mix contains poly-T
(oligo dT) primers, reverse
transcriptase (to make cDNA),
and fluorescently dyed
nucleotides.

We will add cyanine 3 (fluoresces
green) to the healthy cells and
cyanine 5 (fluoresces red) to the
cancerous cells.

The primer and RT bind to the
mRNA first, then add the
fluorescently dyed nucleotides,
creating a complementary strand
of DNA
STEP 4: Hybridization.
 Apply the cDNA we have
just created to a microarray
plate.
 When comparing two
samples, apply both
samples to the same plate.
 The ssDNA will bind to the
cDNA already present on
the plate.
STEP 5:
LASERS!
STEP 5: Microarray Scanner.
 The scanner has a laser, a computer,
and a camera.
 The laser causes the hybrid bonds to
fluoresce.
 The camera records the images
produced when the laser scans the
plate.
 The computer allows us to
immediately view our results and it
also stores our data.
STEP 6:
Analyze the Data.

GREEN – the healthy sample hybridized more
than the diseased sample.

RED – the diseased/cancerous sample
hybridized more than the nondiseased
sample.

YELLOW - both samples hybridized equally to
the target DNA.

BLACK - areas where neither sample
hybridized to the target DNA.
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By comparing the differences in gene
expression between the two samples, we can
understand more about the genomics of a
disease.
Benefits.
 Relatively affordable (for some people!), about $60,000 for an
arrayer and scanner setup.
 The plates are convenient to work with because they are small.
 Fast - Thousands of genes can be analyzed at once.
Problems.
 Oligonucleotide libraries – redundancy and
contamination.
 DNA Microarray only detects whether a gene
is turned on or off.
 Massive amounts of data.
http://www.stuffintheair.com/very-big-problem.html
The Future of DNA Microarray.
 Gene discovery.
 Disease diagnosis: classify the types of cancer on the basis of the
patterns of gene activity in the tumor cells.
 Pharmacogenomics = is the study of correlations between
therapeutic responses to drugs and the genetic profiles of the
patients.
 Toxicogenomics – microarray technology allows us to research the
impact of toxins on cells. Some toxins can change the genetic
profiles of cells, which can be passed on to cell progeny.
Sources.

DNA Microarray Technology. National Human Genome Research Institute, 17 Dec. 2009. 19 Feb.
2010 <http://www.genome.gov/10000533>

Microarrays: Chipping Away at the Mysteries of Science and Medicine. National Center for
Biotechnology Information, 27 July 2007. 19 Feb. 2010.
<http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html>
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Brown, P.O. & Botstein, D. Exploring the New World of the Genome with DNA Microarrays.
Nature Genetics Supplement. 21. (1999): 33-37.
<http://www.ctu.edu.vn/~dvxe/Bioinformatic/PDF%20Files/Volume21/ng0199supp_33.pdf>
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Simon, R., Radmacher, M.D., Dobbin, K., & McShane, L.M. Pitfalls in the Use of DNA Microarray
Data for Diagnostic and Prognostic Classification. Journal of the National Cancer Institute. 95.
(2003): 14-18. http://jnci.oxfordjournals.org/cgi/content/full/95/1/14
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Holloway, A.J., Van Laar, R.K., Tothill, R.W., & Bowtell, D.D.L. Options Available – From Start to
Finish – For Obtaining Data From DNA Microarrays II. Nature Genetics Supplement. 32. (2002):
482-489. <http://web.cs.mun.ca/~harold/Courses/Old/CS6754.W04/Diary/ng1030.pdf>