Microarrays Flashcards
What is a Microarray?
- An ordered assembly of nucleic acids immobilised on a solid support
- Support: Glass like microscope slide
What are Microarray probes?
- 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
What are the features of a microarray?
- 6.5 million locations on each GeneChip array
- Millions of DNA strands built up in each location
- Actual strand = 25 base pairs
How does the detection part of a microarray work?
You shine a laser at the GeneChip array which causes tagged DNA fragments that are hybridised to glow
How does a Microarray work?
View diagram
What have Microarrays been used for in the past?
- Gene expressions (transcriptomics)
- SNP genotyping (SNP arrays)
- Structural variant detection (array CGH)
What are these applications being replaced with now?
Replaced with next generation sequencing protocols:
- RNA sequencing
- Whole Exome - Whole Genome Sequencing (WES/WGS)
What are the expression levels of all genes in your sample?
The Transcriptome
How do you check which genes are expressed at different levels between different types of samples?
- Discover the biology of your sample
- Classify your sample
- Predict which class your sample belongs to
Briefly describe Gene Expression Microarrays
- 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
Describe how expression profiling workflow works
VD
What are the steps in Data analysis workflow?
Feature extraction -> Quality Control -> Normalisation -> Differential Expression analysis -> Biological interpretation -> Submit data to a public repository
Describe the Expression analysis pathway
Normalisation -> Hierarchial Clustering -> Gene Filtering -> Statistical tests -> Generate Gene lists -> Biological Interpretation
What is Clustering?
Organising data with similar patterns into classes. Objects within a class are more similar to each other than objects outside the class
What are data repositories?
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
Describe how qPCR works
cDNA -> RNA -> Protein
This process is also known as reverse transcriptase
How can we make RT-PCR quantitative?
By counting the number of copies of amplified DNA present. We count the copies by using fluorescent molecules - “Tags”
What is the relationship between the RNA (cDNA) and the Ct value?
The higher the amount of starting RNA (cDNA), the lower the Ct value
What techniques are used to count the number of amplified molecules present?
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
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
- 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.
Describe how microarrays are used for SNP genotyping and GWAS
- Genome wide association are only possible because we can genotype large numbers of SNP’s in large numbers of subjects
How is this possible?
- 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
What’s in a spot?
- Lots of copies of the same single stranded oligonucleotide: a probe
- Each probe is for genotyping one SNP
How does it work?
View Diagram
What are the results from genotyping microarrays with SNP?
- Each spot gives the genotype for one SNP
- Upto 5 million spots per sample on array
- Genome wide analysis is possible
What then happens with each probe?
- 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
Watch lecture for the last LO
