MICROARRAY ANALYSIS

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 Gene expression
• There are many different cell types,
but every cell in our body contains
the same genetic information.
• Why?
Gene expression.

Almost every cell in our body contains the same genetic information. Inside the
nucleus of every cell is the same set of chromosomes and genes; only gametes and
red blood cells are unique. Yet there are many different cell types, for example: skin
cells, muscle cells, nerve cells … So, if the genetic information is the same, why are
there so many different cell types? The reason for these differences is gene
expression, which means a gene is switched ‘on’ or ‘off’.

Gene expression refers to activity levels of genes within a cell. Some genes are
expressed (switched on) while others aren’t. This explains why structure and function
of a muscle cell is different from a nerve cell: different genes are expressed. Some
genes are expressed in all cells. These genes are called housekeeping genes as they’re
usually associated with essential cell functions, such as respiration.

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 When are genes expressed?

When a gene is transcribed messenger RNA (mRNA) is produced, and the gene is
expressed. This happens in response to extracellular or intracellular signals. Signals
from inside or outside a cell, trigger gene expression. Different genes are expressed at
different times and in different amounts, depending on environmental conditions and
a cell’s needs. For instance, during wound healing, gene expression of macrophage
inflammatory proteins increases to help clear away infectious material.

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 Measuring gene expression
Different techniques are used to
measure gene expression, such as:
• northern blotting
• reverse transcription polymerase
chain reaction (RT-PCR)
• microarray

The amount of mRNA in a cell provides a measure of gene expression. All these
techniques allow researchers to identify which genes are expressed in a cell at a
particular time, and in what amounts.
Northern blot: this technique detects specific mRNA present in a sample. Researchers
isolate mRNA from cells and separate different sized fragments of mRNA using gel
electrophoresis. These fragments are transferred to a blotting membrane;
radioactively labelled hybridisation probes are added, which bind with the mRNA.
Reverse transcription polymerase chain reaction (RT-PCR): this technique allows for
amplification of mRNA samples and quantification. RT-PCR uses the enzyme, reverse
transcriptase, to convert mRNA into complementary DNA (cDNA). cDNA is then
amplified using PCR and the amounts quantified, allowing expression of one gene at a
time.

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 Microarray and gene expression
• Microarray
technology measures
the amount of mRNA
present in many
samples at once.
• What genes are
expressed and in
what amounts?

Image courtesy of Affymetrix Inc

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Microarray technology is used to investigate gene expression in cells by measuring

the amount of mRNA present in many samples at once. Microarray provides a
‘snapshot’ of which genes are expressed in a cell at a particular moment in time, and
in what amounts.
Microarray is carried out using a gene chip or microarray slide. A gene chip is a solid
surface, usually a glass plate, into which thousands of short strands of single-stranded
DNA are embedded (DNA probes).
DNA strands embedded on microarray slides are from known genes and are
manufactured artificially. Gene chips can contain tens of thousands of genes, and are
used to study expression of thousands of genes simultaneously.

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 Microarray and cancer
Microarray is used to study cancer,
enabling:
• classification of disease subtypes
• identification of genes that may be
useful targets for personalised
medical treatment
• selection of treatments suitable for
individual patients

Cancer is a genetic disease which results in abnormal gene expression. Cancer can
lead to gene expression being overexpressed, underexpressed, and in some cases,
not expressed at all.
Scientists look for differences in gene expression patterns between cancerous cells
and normal cells, for instance, between normal skin cells and melanoma cells.

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 STEPS DURING MICROARRAY ANALYSIS

Step 1: Prepare microarray slide

A microarray slide is embedded with
manufactured single-stranded DNA
segments from known genes.

In this simulation you’ll investigate differences in gene expression between normal

skin cells and melanoma cells.
In microarray experiments there are five main steps.

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Step 2: Collect patient samples
Normal skin cells and melanoma cells
are collected from each patient and
mRNA isolated.

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 Step 3: Convert and label
• mRNA in patient samples is
converted into cDNA.
• Samples are then labelled with
fluorescent dye: normal skin cells
with green dye, and melanoma cells
with red. Once labelled, the two
cDNA samples are mixed together.

mRNA deteriorates easily and isn’t reliable in microarray experiments. To avoid this
problem, an enzyme called reverse transcriptase is used to convert mRNA into
complementary DNA (cDNA). This cDNA is more stable and less likely to degrade
during microarray experiments.

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 Step 4: Add cDNA to microarray slide
• cDNA will hybridise (bind) with
complementary strands embedded
on the microarray slide.

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Microarray works on the principle of complementary base pairing, where guanine

binds with cytosine, and thymine with adenine.
After cDNA has been added the microarray slide is washed to remove any cDNA that
didn’t bind with the DNA probes.

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 Step 5: Visualise microarray results
• Using laser microscopy the
microarray slide is scanned revealing
different coloured spots on the slide:
red, green, yellow and black.

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The colour pattern of the scanned image is analysed to determine which genes are
expressed in each cell type.
Green dot: cDNA from normal skin cells has hybridised with DNA probes. This gene is
expressed in normal cells but not melanoma cells.
Red dot: cDNA from melanoma cells has hybridised with DNA probes. This gene is
expressed in melanoma cells but not normal cells.
Yellow dot: cDNA from both normal skin cells and melanoma cells have hybridised
with DNA probes. Yellow results when both green and red cDNA bind to the same
site.
Black dot: cDNA from neither sample hybridised with DNA probes. This means the
gene is not expressed in either normal skin cells or melanoma cells.
The intensity of fluorescence provides further information about gene expression;
bright red spots indicate higher levels of expression in melanoma cells but not normal
skin cells, and bright green spots indicate higher levels of expression in normal skin
cells but not melanoma cells.

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 Interpreting microarray results

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Powerful computational programmes are required to interpret the data generated by

microarray experiments. Colour spots appearing on a microarray are analysed using
statistical methods.
Each coloured spot on a microarray slide provides information about gene expression,
and helps scientists determine which genes are suitable for investigating in terms of
disease, such as melanoma.
Red and green coloured spots are genes that might be investigated, while yellow and
black spots are probably not involved in cells becoming cancerous.
The range of colours observed in microarray results reflects the relative amount of
mRNA present, or the level of gene expression.

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