Thank you for the summary. This gives a good overview of the key steps and findings from the microarray experiment comparing gene expression in normal vs cancerous cells.

2.  Microarray analysis
 What is DNA microarray ?
 How does a DNA microarray work ?
 What is a DNA microarray used for ?

3.  During the last half of the 20th century, the analysis of the
regulation and function of genes have been largely driven
by step-by-step studies of individual genes and proteins.
 In the past decade, a paradigm shift has emerged in which
we are now able to produce large amounts of data about
many genes in a highly parallel and rapidly serialized
manner.
 An important tool in this process has been the
development of DNA microarray.

4.  The human genome contains approximately up to 40,000
genes. At any given moment, each of our cells has some
combination of these genes turned on, and others are
turned off.
BUT
How do scientists figure out which are on
and which are off?

5.  Scientists can answer this question for any cell sample or
tissue by gene expression profiling, using a technique called
microarray analysis.
 Array definition: to place in an orderly arrangement
 Microarray analysis involves:
◦ Breaking a cell,
◦ Isolating its genetic contents,
◦ Identifying all the genes that are turned on in that particular cell,
◦ Generating a list of those genes

6.  Microarray has become a general term
 There are many types:
• DNA microarrays
• Protein microarrays
• Transfection microarrays
• Antibody microarray
• Tissue microarray
• Chemical compound microarray
..... We’ll be discussing DNA microarrays

7.  DNA microarray analysis is a technique that scientists use
to determine whether genes are on or off.
 Scientists know a gene is on in a cell if its mRNA is present.
 DNA microarray analysis is one of the fastest-growing new
technologies in the field of genetic research.

8.  A DNA microarray allows scientists to perform an experiment on
thousands of genes at the same time.
• Each spot on a microarray contains multiple identical strands of
specific DNA, known as probes (or oligos).
• The DNA sequence on each spot is unique. Each “probe” occupies
a particular “spot” on the chip.
• Each spot represents one gene.
• Thousands of spots are arrayed in orderly rows and columns on a
solid surface (usually glass).
• The precise location and sequence of each spot is recorded in a
computer database.
• Microarrays can be the size of a microscope slide, or even smaller.

9.  A microarray is a pattern of ssDNA probes which are immobilized on
a surface called a chip or a slide.
 Microarrays use hybridization to detect a specific DNA or RNA in a
sample.
 “Fluorescently labeled target sequences” that bind to a “probe
sequence” generate a signal.

12.  When a gene is activated (turned on, expressed) segments
of that gene are copies to give mRNA
 mRNA are body’s template for creating proteins
 To determine turned on or off genes in a given cell, we have
to collect mRNAs of that cell.
 mRNA s are labeled by Reverse Transcriptase (RT) enzyme
that generates cDNA where fluorescent nucleotides are
attached
 Test samples and control ones are labeled differently
 The labeled cDNAs will hybridize to their synthetic
complementary DNAs attached on the microarray slide,
leaving their fluorescent tag
 The fluorescent intensity of each spot are measured.

13. Dr. Flora is studying the effects of high CO2
levels on soybeans. She grew one gp of
soybeans in regular air and another gp of
soybeans in air with high CO2 levels. She
labeled the cDNA from soybeans grown in
high CO2 with red dye and the cDNA from
soybeans grown in normal air with green dye.
Here are the results that she obtained from an
experiment using soybean gene microarray:

14. 1- which spot(s) represent genes that were induced by
elevated CO2?
2- which spot(s) represent genes that do not show a
difference in gene expression in high CO2 level vs
normal air?
3- what does it mean the black color of the spot nb 5?

16.  Gene Discovery: DNA Microarray technology helps in the
identification of new genes, know about their functioning and
expression levels under different conditions.
 Disease Diagnosis: DNA Microarray technology helps
researchers learn more about different diseases such as heart
diseases, mental illness, infectious disease and especially the
study of cancer. Until recently, different types of cancer have
been classified on the basis of the organs in which the tumors
develop. Now, with the evolution of microarray technology, it
will be possible for the researchers to further classify the types
of cancer on the basis of the patterns of gene activity in the
tumor cells. This will tremendously help the pharmaceutical
community to develop more effective drugs as the treatment
strategies will be targeted directly to the specific type of
cancer.

17.  Drug Discovery: Microarray technology has extensive
application in Pharmacogenomics. Pharmacogenomics is the
study of correlations between therapeutic responses to drugs
and the genetic profiles of the patients. Comparative analysis of
the genes from a diseased and a normal cell will help the
identification of the biochemical constitution of the proteins
synthesized by the diseased genes. The researchers can use this
information to synthesize drugs which combat with these
proteins and reduce their effect.
 Toxicological Research: Microarray technology provides a
robust platform for the research of the impact of toxins on the
cells and their passing on to the progeny. Toxicogenomics
establishes correlation between responses to toxicants and the
changes in the genetic profiles of the cells exposed to such
toxicants.

18.  Expression profile: looks at the gene expression
patterns for various diseases, such as cancer.
 Disease screening: genes associated with disease,
early diagnosis and treatment.
 Developmental biology: looks at expression
patterns at different times to identify and
characterize the genes involved.
 Comparative genome hybridization (CGH): a
technique that looks for a genomic gains and
losses or for a change in the number of copies of a
particular gene involved in a disease state.

19. You are a part of a research group studying
human genes involved in cancer. Your group
has decided to use microarrays to compare
gene expression in normal cells vs.
abnormal, cancerous cells. Your goal is to
identify the genes that are expressed
differently. Each research group will work
with 2 different tissue samples: one normal
and one cancerous.

20.  Step 1:
Collect mRNA
 Step 2:
Convert mRNA of each set into labeled cDNA
Ex: 5’-CCUAUUGGAAUCGG-3’
3’-GGATAACCTTAGCC-5’
 Step 3:
Hybridize in the microarray
 Step 4:
Analyze the results

23. Your group has obtained interesting results that may be
useful in determining how cancer cells differ from normal
cells! The next step is to study those genes that appear
to be important in your cancerous cells.
 Which unknown gene sequences (#1–6) appear to belong
to genes used in all cells?
The gene sequences on spots 3 and 5 appear to belong to
genes used in all cells.
 Which unknown gene sequences (#1–6) might belong to
cancer-preventing genes?
The gene sequences on spots 1, 4

24.  Which unknown gene sequences (#1–6) might be from
genes that cause cells to become cancerous?
The gene sequences on spot 6 belong to genes that might
cause cancer.
 Are all of the genes expressed at the same level? How do
you know this?
No, not all genes are expressed at the same level, as
indicated by the fact that different numbers of cDNAs are
bound to the spots representing different genes. The
observation that different genes are expressed at
different levels, especially with respect to the different
levels seen in cancer cells vs. normal cells, suggests that
the level of gene expression is very important in
regulating the cell.