Microarrays Flashcards
What is a microarrays?
An ordered assembly of nucleic acids immobilised on a solid support.
Support – usually glass – like a microscope slide
What are microarray probes?
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
What is the purpose of microarrays?
Microarrays have been widely used for many years now:
- For gene expression (transcriptomics)
- For SNP genotyping (SNP arrays)
- For structural variant detection (array CGH)
Many of these applications are now being replaced with Next Generation Sequencing protocols
- RNA-Seq
- Whole Exome/ Whole Genome sequencing (WES/WGS)
However, not in the clinic just yet!
What do gene expression microarrays show?
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
What is the expression profiling workflow (the steps)?
We have a test sample and control samples
We label one in red and one in green
We create cDNA using reverse transcriptase
We mix the two, hybridise the two to the array
Scan it, get the data and look at the colour
What is the data analysis workflow?
Feature extractions are just getting the data from the array Quality control Normalisation Hierarchical clustering Gene filtering Statistical tests Generate gene list Biological interpretation
What are data repositories?
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
What is quantitive-PCR/qPCR?
A way of checking microarray results
We use RNA to turn it into cDNA (copy DNA) using reverse transcriptase
Then we carry out PCR
We are gonna PCR the gene of interest but also a housekeeping gene
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”.
In qPCR how do you count the number of amplified molecules present?
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
Why is qPCR used?
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
What are SNP microarrays?
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
What’s in a microarray spot?
Lots of copies of the same single-stranded oligonucleotide – a “probe”
Each probe is for genotyping one SNP
Each spot gives the genotype for one SNP
Up to 5 million spots per sample on array
Genome-wide analysis possible
What is Array Comparative Genomic Hybridisation (aCGH)?
A laboratory technique used to look for alternations in chromosomes that are too small to see down a microscope
Using an array we mix labelled patient and control DNA we hybridise it to an array, scan it and for the vast majority there will be equal spots.
Some areas with more green when there has been a duplication
More red means there has been a deletion of bases in the patient
