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?
 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.

 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>

 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>

 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

 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>