Transcript Microarray

Microarray: An Introduction
Gene Background
• Gene: a region of the human genome
coding for a protein
• Biologists interested in gene function and
disease
• Studying multiple genes is time consuming
and expensive
§ Genes are differentially expressed in...
• Different cell types (e.g. muscle cells, fibroblasts)
• Environmental conditions (e.g. heat shock,
nutrient deprivation)
• Developmental phases
• Cell-cycle stages (e.g. G1 phase)
• Disease states (e.g. tumor cells, virus-infected
cells)
• Gene expression is primarily regulated
at the level of transcription
§ Hence, the number of mRNA copies in a
cell for a particular gene is a good
indicator of that gene’s expression
(number of proteins)
§ Dynamic range of mRNA levels:
• Highly expressed genes can have up to
9400 mRNA copies per cell
• Poorly expressed genes can have < 0.3
mRNA copies per cell
Regulation of Gene Expression
m-RNA
stability
Control
RNA
process
control
NUCLEAS
Translation
control
Cytosol
DNA
Transcriptional
Control
Primary
RNA
transcript
m-RNA
m-RNA
Protein
Active
protein
Transport
Control
Posttranslational
Control
Traditional Methods
• Northern Blotting
– Single RNA isolated
– Probed with labeled cDNA
• Reverse Transcription – Polymerase
Chain Reaction (RT-PCR)
• Real time or quantitative RT-PCR
– Each technique measures the m-RNA levels
of one or a few genes at a time
Microarray Technology
• Microarray:
– New Technology (first paper: 1995)
• Allows study of thousands of genes at same time
• Tools used to measure simultaneously the
transcription level of every gene in a cell
Advantages of DNA microarray
experiments
• Lack information a priori of which genes may
have altered mRNA levels in a particular
experimental condition
• Knowledge of patterns of genes with altered
mRNA levels is usually more informative than
knowledge of single genes with altered mRNA
levels
• Use statistical analysis of microarray data to test
hypotheses or generate new hypotheses
Microarray Technology
Quantitative Measurement of
Gene Expression
• Also known as DNA microarrays, DNA arrays, DNA chips,
gene chips, … Whatever the name, their use is effectively
transforming a living from a black box into a transparent box.
Applications of Microarray
Technology
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Applications of Microarray
Technology
• What type of data analysis is required to:
– Identify Genes expressed in different cell types (e.g. Liver
vs finger)
– Learn how expression levels change in different
developmental stages (embryo vs. adult)
– Learn how expression levels change in different
developmental stages (cancerous vs non-cancerous)
– Learn how groups of genes inter-relate (gene-gene
interactions)
– Identify cellular processes that genes participate in
(structure, repair, metabolism, replication, … etc)
• Applications covered only as example contexts,
emphasis is on analysis methods
Microarray Technology
• Types of
microarray
technologies and
how they work
• Outputs of
microarrays
• Image Analysis
required to
transform output
to gene
expression
matrices
Fabrications of Microarrays
• Size of a microscope slide
Images: http://www.affymetrix.com/
The Mechanics of a DNA
Microarray Experiment
• Isolate mRNA from cell cultures
• Reverse Transcribe mRNA into cDNA
• Label cDNA or cRNA by incorporating
fluorescently-labeled nucleotides
• Hybridize labeled cDNA (or cRNA) to DNA
microarray
• Wash and scan microarray in confocal
laser scanner
• Analyze data
Differing Conditions
• Ultimate Goal:
– Understand expression level of genes under
different conditions
• Helps to:
– Determine genes involved in a disease
– Pathways to a disease
– Used as a screening tool
Gene Conditions
•
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Cell types (brain vs. liver)
Developmental (fetal vs. adult)
Response to stimulus
Gene activity (wild vs. mutant)
Disease states (healthy vs. diseased)
Expressed Genes
• Genes under a given condition
– mRNA extracted from cells
– mRNA labeled
– Labeled mRNA is mRNA present in a given
condition
– Labeled mRNA will hybridize (base pair) with
corresponding sequence on slide
Constructing DNA Microarrays
• A DNA microarray is a collection of DNA probes
separated in a regular array atop a solid support
(glass slide, silicon chip, etc)
• Affymetrix oligonucleotide microarrays
Short (25mers) oligonucleotide probes synthesized
on the array
• cDNA microarrays
cDNA PCR products printed on coated glass slides
• Oligonucleotide microarrays
Long (70 - 100mers) oligonucleotide probes are
printed on coated glass slides
Custom Arrays
• Mostly cDNA arrays
• 2-dye (2-channel)
– RNA from two sources (cDNA created)
• Source 1: labeled with red dye (Cy5)
• Source 2: labeled with green dye (Cy3)
Two Channel Microarrays
• Microarrays measure gene expression
• Two different samples:
– Control (green label)
– Sample (red label)
• Both are washed over the microarray
– Hybridization occurs
– Each spot is one of 4 colors
Building the chip
Ngai Lab arrayer , UC Berkeley
Print-tip head
Pins collect cDNA
from wells
well plate
Contains cDNA
probes
Print-tip
group 1
cDNA clones
Glass Slide
Array of bound cDNA probes
In this case:
4x4 blocks = 16 print-tip groups
Print-tip
group 7
Microarray Image Analysis
• Microarrays detect gene
interactions: 4 colors:
–
–
–
–
Green: high control
Red: High sample
Yellow: Equal
Black: None
• Problem is to quantify
image signals
A two-channel microarray
experiment
Microarray Animations
• Davidson University:
• http://www.bio.davidson.edu/courses/genomics/chip/chip.html
• Imagecyte:
• http://www.imagecyte.com/array2.html