Microarrays

Question 1

What is a Microarray?

Answer 1

• An ordered assembly of nucleic acids immobilised on a solid support
• Support: Glass like microscope slide

Question 2

What are Microarray probes?

Answer 2

• Probes are 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

What are the features of a microarray?

Answer 3

• 6.5 million locations on each GeneChip array
• Millions of DNA strands built up in each location
• Actual strand = 25 base pairs

Question 4

How does the detection part of a microarray work?

Answer 4

You shine a laser at the GeneChip array which causes tagged DNA fragments that are hybridised to glow

Question 5

How does a Microarray work?

Answer 5

View diagram

Question 6

What have Microarrays been used for in the past?

Answer 6

• Gene expressions (transcriptomics)
• SNP genotyping (SNP arrays)
• Structural variant detection (array CGH)

Question 7

What are these applications being replaced with now?

Answer 7

Replaced with next generation sequencing protocols:
- RNA sequencing
- Whole Exome - Whole Genome Sequencing (WES/WGS)

Question 8

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

Answer 8

The Transcriptome

Question 9

How do you check which genes are expressed at different levels between different types of samples?

Answer 9

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

Question 10

Briefly describe Gene Expression Microarrays

Answer 10

• 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

Question 11

Describe how expression profiling workflow works

Answer 11

VD

Question 12

What are the steps in Data analysis workflow?

Answer 12

Feature extraction -> Quality Control -> Normalisation -> Differential Expression analysis -> Biological interpretation -> Submit data to a public repository

Question 13

Describe the Expression analysis pathway

Answer 13

Normalisation -> Hierarchial Clustering -> Gene Filtering -> Statistical tests -> Generate Gene lists -> Biological Interpretation

Question 14

What is Clustering?

Answer 14

Organising data with similar patterns into classes. Objects within a class are more similar to each other than objects outside the class

Question 15

What are data repositories?

Answer 15

They maximise utility of microarray experiments
Share data / Use other people’s data

• If users provide the minimum information about a microarray experiment then it’s easier to compare results
• ArrayExpress, EBI
• GEO: Gene Expression Omnibus, NCBI

Question 16

Describe how qPCR works

Answer 16

cDNA -> RNA -> Protein
This process is also known as reverse transcriptase

Question 17

How can we make RT-PCR quantitative?

Answer 17

By counting the number of copies of amplified DNA present. We count the copies by using fluorescent molecules - “Tags”

Question 18

What is the relationship between the RNA (cDNA) and the Ct value?

Answer 18

The higher the amount of starting RNA (cDNA), the lower the Ct value

Question 19

What techniques are used to count the number of amplified molecules present?

Answer 19

1). Include a dye in the PCR reaction mix that fluoresces when it binds double stranded DNA
Or
2). Label a probe in the PCR that only fluoresces when it is incorporated in the PCR product

Question 20

Why is qPCR used?

Answer 20

• 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
• RNA-seq is a more accurate measure of RNA transcript abundance, it’s more reproducible and works over a much wider range of concentrations but is more expensive.

Question 21

Describe how microarrays are used for SNP genotyping and GWAS

Answer 21

• Genome wide association are only possible because we can genotype large numbers of SNP’s in large numbers of subjects

Question 22

How is this possible?

Answer 22

• Possible by using microarrays that hybridise with genomic DNA adjacent to SNP’s (rather than RNA transcripts)
• The SNP is then extended by one base that is fluoresently labelled and detected using a high definition scanner

Question 23

What’s in a spot?

Answer 23

• Lots of copies of the same single stranded oligonucleotide: a probe
• Each probe is for genotyping one SNP

Question 24

How does it work?

Answer 24

View Diagram

Question 25

What are the results from genotyping microarrays with SNP?

Answer 25

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

Question 26

What then happens with each probe?

Answer 26

• The Software translates the three different colour signals for each probe into genotype
• A few SNP’s are reviewed by hand (less than 50) but most are not

Question 27

Watch lecture for the last LO