Microarrays Flashcards
What are microarrays?
Microarrays are an ordered assembly of nucleic acids immobilised on a solid support (this would be glass, similar to a microscope slide).
What are transcriptomics, and how is it used?
Transcriptomics is the study of all the RNA molecules within a cell.
You could use it to look at the expression levels of all genes in the samples; it could be of interest if there are genes that are up or down-regulated in a diseased cell.
You can use it to discover the biology of your samples, classify them, and then predict which class a sample belongs to.
How are microarrays used to calculate gene expression?
When we scan the microarray slide, we end up with an image containing red, green and yellow spots. Each spot represents one SNP.
Because we have lots of spots, we can analyse lots of spots simultaneously. Therefore, microarrays allow us to analyse genetic markers across the genome.
Describe the profiling workflow.
- first, you would extract a sample
- from your total RNA, you would purify out the message (so remove rRNA and tRNA, etc.) and label what’s left
- you would then hybridise the labelled sample to the array
- you would the scan the chip with a laser to detect the signal
- you would then perform data analysis, including analysis and biological interpretation
What do you do after you obtain the raw data from the microarray?
- NORMALISATION: this is making sure that there are no other reasons why the probes are binding to the samples other than the fact that they’re being expressed (some probes are known to bind non-specifically - there are lots of control arrays to try and stop this)
- HIERARCHICAL CLUSTERING: the core technique
- GENE FILTERING: you filter out the genes that you aren’t interested in
- STATISTICAL TESTS: you perform statistical tests on the data to determine significance
- GENERATE GENE LIST: where you create a list of interesting findings using pathways analysis (this is where you take all the genes and show which ones are up/down-regulated or don’t change in our disease or biological trait)
- BIOLOGICAL INTERPRETATION: you would then impose biological interpretation on the results
Describe how clustering works.
It organises the data with similar patterns into classes. Objects within a class are more similar to each other than to objects outside the class.
Your clustering technique might give you the big cluster (the whole thing) or the smaller clusters, so interpretation can be challenging.
Another way in which clustering is presented is by dendograms (‘trees’). The distant sample are less similar, and vice versa.
How did data repositories come about?
Microarray experiments aren’t cheap, so to maximise the utility we share the data and use other people’s data.
If users provide the minimum information about a microarray experiment (MIAME) then it is easier to compare results, so they all follow this criteria.
An example of some of them is:
- ArrayExpress
- EBI (European Bioinformatic Institute)
- GEO (Gene Expression Omnibus)
- NCBI (National Centre for Biotechnology Information)
How are microarrays used in the NHS?
The NHS uses microarrays for breast cancer management and chemotherapy decisions.
Some gene expression profiling and expanded immunohistochemistry tests (used for guiding adjuvant chemotherapy decisions in early breast cancer management):
- MammaPrint
- Oncotype DX
- IHC4
- Mammostrat
Why would we do reverse transcription PCR?
We would do it to see if certain elements are being expressed or not.
The real question would be if we can quantify it, which is when we would use Q-PCR.
How would we make RT-PCR quantitative?
We could do so by counting the number of copies of amplified DNA present.
We count the copies by using fluorescent molecules - ‘tags’.
What is the Ct value in Q-PCR?
The Ct value is the fluorescence above background at 225 copies. It is an arbitrary value that has been chosen to allow you to compare across all experiments.
The higher the amount of starting RNA (cRNA), the lower the Ct value.
How do you count the number of amplified molecules present?
You could either:
- 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 would you use quantitative-PCR?
- 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 wider range of concentrations, but it is more expensive
How do SNP microarrays happen?
Genome-wide Association Studies (GWAS) 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 immobilised piece of DNA on the microarray is complementary to the DNA just before the SNP (a single base before).
The SNP is then extended by one base that is fluorescently labelled and detected using a high definition scanner.
What’s in a spot?
In each spot, there are lots of copies of the same single-stranded oligonucleotide – a “probe”
Each probe is for genotyping one SNP.
