Learning Outcomes

High Throughput Gene Expression Analysis:

Microarrays
Analyze the microarray approach to gene expression analysis,
including both the theory and methods behind gene chip production,
mobile probe preparation, experimental design, and results analysis.
Outline the steps in a protein expression profiling experiment,
explain its benefits in a diagnostic setting, and outline its advantages
over a gene expression assay.
Simulate a gene-expression microarray experiment and successfully
interpret results.
 mRNA
mRNA
No mRNA or
protein produced

No mRNA or
protein produced
 The Transcriptome
defined as the complete set of transcripts in an organism
under defined condititons
can be analyzed as a whole (thousands of genes at
once) using the following technologies:
 microarrays (cDNA and oligonucleotide)
comparing relative levels of gene expression across
tissues/conditions
 Gene expression
 has an "on/off" switch to control which genes
are expressed in a cell
 has a "volume control" that increases or
decreases the level of expression of particular
genes as necessary

Gene expression from Gene expression from

Obese Tissue Sample Non-Obese Tissue Sample

ON ON OFF ON OFF ON

mRNA mRNA mRNA mRNA

 DNA Microarrays: a high-
throughput technology used to
study gene expression
Step #1: Make the DNA Microarray http://www.agilent.com/about/newsroom/lsca/
imagelibrary/images/
cag_42_microarray_dna_print.jpg

• put tiny amount of single-stranded DNA corresponding to each of one of

thousands of genes onto a microscope-slide (spotted array)
• Can also synthesize the DNA strands using known sequences directly on the
microarray (oligonucleotide array)
• Each spot contains multiple, identical, single-stranded copies of a particular
gene
• The identity and microarray location of each gene is known (this is the
immobilized target DNA**)
Please view the following videos to see how gene chips (microarrays)
are made.
Making Gene Chips (oligonucleotide arrays)
Microarray manufacturing technology (spotted arrays)
 Step #2: Prepare the mobile probe(s)
Reminder: gene expression is measured by the presence/absence and
abundance of mRNA in a cell

• Isolate mRNA from desired samples (i.e. fat cells from obese and non-obese
individuals)
• Make fluorescently-labeled single-stranded DNA copies (cDNA)
• These probes represent every gene that is turned “ON” in your sample

Gene expression from Gene expression from

Obese Tissue Sample Non-Obese Tissue Sample
ON ON OFF ON OFF ON

mRNA mRNA mRNA mRNA

cDNA
cDNA
cDNA cDNA
Generating cDNA: first isolate mRNA

Isolate total RNA from eukaryotic

cells
mRNAs contain a poly A tail
Use this feature to purify the
mRNA from the more abundant
tRNA and rRNA: bind mRNA to
solid matrix containing short
stretches of thymidine residues
(olido dT column)

Lodish et al; Fig 7-14

Generating cDNA

All cDNA molecules

from one sample will
be fluorescently
labeled with one
colour
 Step #3: Hybridization Probing
• Add single-stranded probe(s) to microarray
• when two complementary sequences find each other they will lock together, or
hybridize.
• You can use one microarray to determine the difference in gene expression between
2 samples
• generally 2 samples (each labeled with a unique colour) are added to each array to
compare the relative level of expression of each gene (E.g. WT & mutant, normal vs.
diseased sample)
• Wash step!!

http://learn.genetics.utah.edu/content/labs/microarray/analysis/ Image credit: Affymetrix

Step #4: Microarray Scanner

• Insert the GeneChip

(microarray) into a microarray
scanner (computer)
• Detect colour/intensity of
fluorescence

• amount of probe that binds to

each spot is proportional to
the abundance of the specific
transcript in the cell or tissue
sample
http://learn.genetics.utah.edu/content/labs/microarray/analysis/
Level of expression:
Genes that areRemember: the identity of an abundance of mRNA=very bright
•
highly expressed produce
signals each microarray location is
known
Genes that are moderately expressed produce fewer mRNAs=dimmer signals
Genes that are “OFF” don’t produce any mRNA=no signal detected
 **If you have used 2 different probes (from 2 different samples), you will
end up with “colour mixing” of the fluorescent probes. Please see the
following slide.

http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
 Visions of DNA Microarrays

http://www.npaci.edu/online/v4.12/pettitt.html

Microarray Animation
-watch this animation for a good
review of microarrays
 Microarray Analysis

just seeing a transcript does not guarantee that the protein is produced or
functional
Usually the comparison of gene expression profiles is of most interest
If a difference in transcript abundance is observed between two or more
conditions, it might point to an interesting biological phenomenon

Requires appropriate replication, normalization, and statistics

output of all microarray experiments is ultimately a series of numbers,

which can cover a range of almost four orders of magnitude,(I.e from
perhaps one transcript per ten cells to a few thousand transcripts per cell)
 Biological Expression Profiles

Scientists can:
Use DNA microarrays to study gene expression profiles
Analyze protein expression to determine which proteins are
functional in a specific cell or tissue

Question:
If we can easily determine gene expression status, why would
a scientist want to study protein expression?
 Biological Expression Profiles

Scientists can:
Use DNA microarrays to study gene expression profiles
Analyze protein expression to determine which proteins are
functional in a specific cell or tissue

Question:
If we can easily determine gene expression status, why would
a scientist want to study protein expression?

Answer:
Even if a gene is turned on, there is no guarantee that the
protein it encodes is being made (just because you have
mRNA doesn’t mean you have protein).
 DNA Microarray Applications

Gene expression profiling

assay gene expression within a single sample
(which genes are on/off)
compare gene expression in two different cell
types/tissue samples/or at two different times
(i.e. healthy versus diseased tissue)
SNP profiling
Identify single nucleotide polymorphisms in an
individual
Compare SNP patterns in diseased and normal cells
http://learn.genetics.utah.edu/units/pharma/phprotan/
Examples of microarray experiments
1. Tissue-specific gene expression
identify which genes are preferentially expressed in which tissues.

2. Developmental genetics
Different genes express at different stages of its developmental
process.
Microarrays can track the changes in developmental gene expression
profiles

3. Genetic diseases:
Identify which genes are differentially expressed in diseased Vs
normal cells.
Development of drugs that are specific to the difference between
diseased Vs normal

4. Complex diseases
Identify the genetic markers that may predispose an individual to a
complex disease.
5. Pharmacological agents
Gene expression patterns maybe altered by external stimulus:
pharmacological agent
Identify those genes which express differently in response to such
exposure.
You should have already
completed this self-directed
learning module:
Visit the following website and
complete the virtual microarray
lab. Work carefully through all
three chapters
http://learn.genetics.utah.edu/co
ntent/labs/microarray/
 Microarray Wet-Lab Simulation

How to analyze microarray data (optional)

http://www.hhmi.org/biointeractive/how-analyze-d
na-microarray-data

Microarrays review:
https://www.youtube.com/watch?v=0ATUjAxNf6U