Chips & Microarrays

Question 1

What is a microarray?

Answer 1

A tool for analysing gene expression
Small membrane / glass slide containing samples of thousands of genes arranged in a pattern

Question 2

Which type of sample does a microarray measure?

Answer 2

RNA
Gives insights into gene expression patterns or changes

Question 3

When are microarrays used in gene expression profiling?

Answer 3

In different cells or tissues
During the course of development
Under different environmental or chemical stimuli
In disease states

Question 4

When are microarrays used in molecular diagnosis?

Answer 4

To molecularly classify diseases

Question 5

When are microarrays used in drug development?

Answer 5

To identify new drug targets

Question 6

When are microarrays used in pharmacogenomics?

Answer 6

To develop individualised medicine

Question 7

What are the types of microarrays?

Answer 7

Spotted DNA arrays (cDNA arrays)
Oligonucleotide arrays (Affymetrix gene chips)
Ink-jet microarrays (Agilent)

Question 8

What are spotted DNA arrays?

Answer 8

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

Question 9

What are oligonucleotide arrays?

Answer 9

Large number of 20-25mers/gene
Enabled by photolithography from the computer industry

Question 10

What are ink-jet microarrays?

Answer 10

25-60mers “synthesised” directly on glass
Four cartridges: A, C, G, and T

Question 11

What are the potential challenges of experimentally designing a microarray?

Answer 11

Hypothesis testing vs. exploratory “fishing expedition”
Errors in approach

Question 12

What are the potential challenges of statistically analysing a microarray?

Answer 12

Small number of samples compared to large number of variables (genes)
Problems with false positives

Question 13

What are the potential challenges of data mining a microarray?

Answer 13

Annotated lists
Function of the differentially expressed genes
Extensive use of online resources

All could be sources of random or systematic measurement errors

Question 14

How and why is DNA/RNA hybridisation used?

Answer 14

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

Question 15

How are samples labelled before being applied to an array?

Answer 15

Using a fluorescent dye

Question 16

How do you read the output of a microarray?

Answer 16

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

Question 17

What is the process of microarray attachment and hybridisation?

Answer 17

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

Question 18

Why would dual labelling be used for some arrays?

Answer 18

Allows the simultaneous measurement of 2 samples- 1 labelled with each dye
e.g., differential analysis, disease vs. normal

Question 19

How do you interpret results of dual label (green, red) experiments?

Answer 19

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

Question 20

How do you acquire data from the qualitative results of a microarray?

Answer 20

Scan the arrays
Quantitate each spot
Subtract background
Normalize
Export a table of fluorescent intensities for each gene in the array

Question 21

How do you analyse array data?

Answer 21

Divide the expression measurement of each probe by the median
Log2 of the expression ratio

Question 22

What are the advantages and disadvantages of median centering?

Answer 22

Median is more robust to outliers than the mean
Assumes the majority of genes are un-changed between conditions

Question 23

What is false discovery rate correction?

Answer 23

Assumption that 5% of results (p = 0.05) will be false negatives
Conservative estimation of differences in gene expression using p = 0.05

Question 24

How are the large number of variables (genes) in microarrays overcome in analysis?

Answer 24

Principal component analysis (gene ontology/gene set enrichment)
Pathway-based analysis