Microarrays

Question 1

What is a microarray?

Answer 1

n ordered assembly of nucleic acids immobilised on a solid support.
• Support – glass – like a microscope slide

Question 2

Microarray probes

Answer 2

• Probes are the short pieces of single-stranded DNA immobilised on the surface of the array
• They are oligonucleotides
• Each spot on the array consists of thousands of probes with the same sequence

Question 3

Quantitive -PCR

Answer 3

We can make RT-PCR quantitative by counting the number of copies of amplified DNA present.

We count the copies by using fluorescent molecules - “tags”.

Question 4

How do you count the number of amplified molecules present?

Answer 4

Include a dye in the PCR reaction mix that fluoresces when it binds double-stranded DNA, e.g. an intercalating dye such as SYBR Green
Or
Label a probe in the PCR that only fluoresces when it is incorporated in the PCR product, e.g. TaqMan

Question 5

Why qPCR?

Answer 5

• qPCR is used to independently confirm differences in RNA levels between samples
• Probe binding is noisy and differences can be detected that are not real, especially where differences are small (<2-fold)
• RNA-Seq is a more accurate measure of RNA transcript abundance, it is more reproducible and works over a much wider range of concentrations…..but it is more expensive

Question 6

Endopredict

Answer 6

Company - Myriad genetics inc.

Technique - 12 genes qPCR

Question 7

Oncotype Dx

Answer 7

Company - Genomic health

Technique - 21 genes - qPCR

Question 8

Prosgng Gene signature

Answer 8

Company - Nanotring

Technique - 21 genes qPCR

Question 9

Breast Cancer Endopredict Risk Estimation

Answer 9

• At a low score (EPclin<3), endocrine therapy (ET) alone is sufficient
• At higher scores, ET+C is clearly beneficial compared to ET alone
• This test ensures that only patients who will benefit from chemotherapy will receive it

Question 10

What are the expression levels of all genes in my sample?

Answer 10

The transcriptome

Question 11

Gene expression microarrays

Answer 11

• Lots of copies of the same probe in a spot
• Each spot gives the relative expression for one transcript
• Detects all known transcripts in one sample
• Each spot represents one SNP
• Because we have lots of spots we can analyse lots of spots simultaneously
• And microarrays therefore allow us to analyse genetic markers across the genome

Question 12

Which genes are expressed at different levels between two different types of samples?

Answer 12

• Discover the biology of your samples
• Classify samples
• Predict which class a sample belongs to

Question 13

Gene expression microarrays

Answer 13

• Lots of copies of the same probe in a spot
• Each spot gives the relative expression for one transcript
• Detects all known transcripts in one sample
• Each spot represents one SNP
• Because we have lots of spots we can analyse lots of spots simultaneously
• And microarrays therefore allow us to analyse genetic markers across the genome

Question 14

Expression Analysis

Answer 14

• Normalisation
• Hierarchical Clustering
• Gene filtering
• Statistical Tests
• Generate gene list
• Biological interpretation

Question 15

Clustering

Answer 15

• Organises data with similar patterns into classes

* Objects within a class are more similar to each other than to objects outside the class

Question 16

Dendrograms

Answer 16

Trees
Alternative way of displaying similarity between samples
Distant samples are less similar

Question 17

Data repositories

Answer 17

• Maximise utility of microarray experiments
- share data
- use other people’s data
• If users provide the Minimum Information About a Microarray Experiment (MIAME) then it is easier to compare results
• ArrayExpress, EBI (European Bioinformatics Institute)
• GEO – Gene Expression Omnibus, NCBI (National Center for Biotechnology Information, USA)

Question 18

SNP microarrays

Answer 18

• Genome-wide Association Studies are only possible because we can genotype large numbers of SNPs in large numbers of subjects
• This is possible by using microarrays that hybridise with genomic DNA adjacent to SNPs (rather than RNA transcripts)
• The SNP is then extended by one base that is fluorescently labelled and detected using a high definition scanner

Question 19

What in a spot

Answer 19

• Lots of copies of the same single-stranded oligonucleotide – a “probe”
• Each probe is for genotyping one SNP

Question 20

Genotyping microarrys

Answer 20

• Each spot gives the genotype for one SNP
• Up to 5 million spots per sample on array
• Genome-wide analysis possible

Question 21

SNP Genotype

Answer 21

• Software translates the three different colour signals for each probe into genotypes
• A few SNPs are reviewed by hand (<50) but most are not