Chips & Microarrays Flashcards
What is a microarray?
A tool for analysing gene expression
Small membrane / glass slide containing samples of thousands of genes arranged in a pattern
Which type of sample does a microarray measure?
RNA
Gives insights into gene expression patterns or changes
When are microarrays used in gene expression profiling?
In different cells or tissues
During the course of development
Under different environmental or chemical stimuli
In disease states
When are microarrays used in molecular diagnosis?
To molecularly classify diseases
When are microarrays used in drug development?
To identify new drug targets
When are microarrays used in pharmacogenomics?
To develop individualised medicine
What are the types of microarrays?
Spotted DNA arrays (cDNA arrays)
Oligonucleotide arrays (Affymetrix gene chips)
Ink-jet microarrays (Agilent)
What are spotted DNA arrays?
PCR products (or long oligonucleotides) from known genes (~100 nt) spotted on glass, plastic, or nylon support
Customizable or off the shelf options
Uses a robot with many pins which are dipped into the PCR products/oligos and then spotted onto the slides
What are oligonucleotide arrays?
Large number of 20-25mers/gene
Enabled by photolithography from the computer industry
What are ink-jet microarrays?
25-60mers “synthesised” directly on glass
Four cartridges: A, C, G, and T
What are the potential challenges of experimentally designing a microarray?
Hypothesis testing vs. exploratory “fishing expedition”
Errors in approach
What are the potential challenges of statistically analysing a microarray?
Small number of samples compared to large number of variables (genes)
Problems with false positives
What are the potential challenges of data mining a microarray?
Annotated lists
Function of the differentially expressed genes
Extensive use of online resources
All could be sources of random or systematic measurement errors
How and why is DNA/RNA hybridisation used?
DNA and RNA is heated to denaturation temperatures to form single strands and then cooled
RNA hybridizes with DNA forming a complementary double helix
A DNA/RNA hybrid is more stable than DNA/DNA hybrid
How are samples labelled before being applied to an array?
Using a fluorescent dye
How do you read the output of a microarray?
Laser light is applied to the array
The fluorescent label enables the detection of which probes have hybridised (presence) via the light emitted from the probe
What is the process of microarray attachment and hybridisation?
Surface chemistry is used to attach the probe molecules to the glass substrate
Chemical reactions are used to attach the florescent dyes to the target molecules
Probe and target hybridise to form a double helix
Why would dual labelling be used for some arrays?
Allows the simultaneous measurement of 2 samples- 1 labelled with each dye
e.g., differential analysis, disease vs. normal
How do you interpret results of dual label (green, red) experiments?
GREEN represents high control hybridization
RED represents high sample hybridization
YELLOW represents a combination of control and sample where both hybridized equally
BLACK represents areas where neither the control nor sample hybridized
How do you acquire data from the qualitative results of a microarray?
Scan the arrays
Quantitate each spot
Subtract background
Normalize
Export a table of fluorescent intensities for each gene in the array
How do you analyse array data?
Divide the expression measurement of each probe by the median
Log2 of the expression ratio
What are the advantages and disadvantages of median centering?
Median is more robust to outliers than the mean
Assumes the majority of genes are un-changed between conditions
What is false discovery rate correction?
Assumption that 5% of results (p = 0.05) will be false negatives
Conservative estimation of differences in gene expression using p = 0.05
How are the large number of variables (genes) in microarrays overcome in analysis?
Principal component analysis (gene ontology/gene set enrichment)
Pathway-based analysis
