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Transcript
Microarrays:
Tools & Interpretation of Data
Kirsty Jensen
Microarrays
• What is a microarray?
– How the technology has evolved
• Macroarrays
• Glass microarrays
• Affymetrix
• Designing a microarray experiment
– Choice of arrays
– Experimental design
– RNA samples
• Analysis
–
–
–
–
–
–
Scanning
Quantification of spots
Normalization
Statistical analysis
Identification of genes of interest
Validation
What Is A Microarray?
“A microarray is a tool for
analyzing gene expression that
consists of a small membrane or
glass slide containing samples of
many genes arranged in a regular
pattern.”
Uses
•
•
•
•
•
Analysis of gene expression
SNP analysis
MHC typing
Identification of pathogens
Identification of tumours
Microarrays
• The underlying principle of microarray
technology is the ability of DNA to bind
to itself and to RNA.
• Analyzing gene expression involves
the detection of mRNA species
(transcriptome) present in a cell or
tissue at a particular point in time.
Evolution of Microarrays
• One gene / one experiment
– Northern blots, RNase assays, RT-PCR
– Hypothesis driven
• Macroarrays
– Nylon membranes
– 32P labelled probes
Macroarrays
RNA
RNA
32P labelled
32P labelled
cDNA
cDNA
Hybridization
Hybridization
Evolution of Microarrays
• One gene / one experiment
– RT-PCR, northern blots, RNase assays
– Hypothesis driven
• Macroarrays
– Nylon membranes
– 32P labelled samples
• Microarrays
– Glass slides
– Fluorescent labelled samples
– Two samples per microarray
Microarrays
RNA
RNA
Cy3 labelled
cDNA
Cy5 labelled
cDNA
Hybridization
Glass Microarrays
• cDNA arrays
– >1000 nucleotides in length
– cDNA libraries
• Oligonucleotide arrays
– 70-80 nucleotides in length
– Increased specificity
Evolution of Microarrays
• One gene / one experiment
– RT-PCR, northern blots, RNase assays
– Hypothesis driven
• Macroarrays
– Nylon membranes
– 32P labelled samples
• Microarrays
– Glass slides
– Fluorescent labelled samples
• Affymetrix
Affymetrix
• 25 nucleotides in length
• Mismatch pair
– Single mismatch at centre of probe
– Used to detect & eliminate false results
• 11 probe pairs per gene
• Photolithography
Affymetrix - Construction
Affymetrix
Affymetrix
RNA
cDNA
Biotin-cRNA
Hybridization
Streptavidin-phycoerythrin
Bovine Microarrays
Microarray
Format
Source of Material
BOTL
cDNA
Bovine total leukocyte cDNA library & immune
gene amplicons
Pyxis CattleArray 7600
cDNA
Bovine spleen & placental cDNA libraries
NBFGC
cDNA
MARC & BARC cDNA libraries
BMAM
cDNA
MARC & BARC cDNA libraries
Mammary gland specific
Endometrial/placental
microarray
cDNA
Bovine placental & endometrial cDNA libraries
Bovine immuneendocrine microarray
cDNA
Bovine immune & endocrine gene amplicons
Bovine MHC class I
microarray
oligos
BoLA DRB3 & DQA sequences
BoMP microarray
RIGUA
cDNA
Oligos
Bovine macrophage cDNA library
Bovine immune & inflammatory gene amplicons
Affymetrix
oligos
Various cDNA libraries
ARK-Genomics
cDNA
Various cDNA libraries
Conclusions (1)
1. Microarrays have many uses.
2. Major use is to investigate gene expression.
3. mRNA isolated from a cell gives a snapshot of the events occurring at that time.
4. There are 3 main types of microarrays
• Glass cDNA microarrays
• Glass oligonucleotide microarrays
• Affymetrix