W3 - Microarrays Flashcards
What is a microarray?
And ordered assembly of nucleic acids immobilised on a solid support.
Support - glass - like a microscope slide
If you look at it closely with a microscope and illuminate it with a scanner, will be able to see multicoloured spots. Each colour corresponds to measurements of a specific DNA hybridisation.
What does the array compose of?
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
How does a Microarray work?
They each have 6 1/2 million locations on each array.
On each location, we have millions of DNA strands built up all with the same sequence (single stranded).
If we did in a DNA fragments that were tagged with fluorescent molecules, anything that they are complementary to will hybridise to correctly.
When you shine a laser light on them, the fluorescence tag will glow. This will be visible through the microscope or scanner or detector. If nothing hybridises, no glow.
How does gene expression microarray work?
What are the expression levels of all genes in my samples?
The transcriptome - combination of all RNA
Which genes are expressed at different levels between two different types of samples?
-Discover the biology of your samples
-Classify samples
-Predict which class a sample belongs to
- lots of copies of the same probe on the spot.
- each spot gives the relative expression for one transcript
- detects all known transcripts in one sample
How does expression profiling workflow work?
one colour and two colour array
How does the two colour array work?
We have a test sample and our control sample.
The isolate the RNA and make mRNA
We label one in red = Cy5
And the control in green = Cy3
We create cDNA using reverse transcriptase.
We mix the two and hybridise them.
We scan it and get the data
We look at the relative colour.
Roughly the same = yellow
If test use stronger it will be more red vise versa.
What steps are involved in data analysis workflow?
feature extraction - getting the raw data from array (CEL file)
Quality control - any issues about concentration or quality of the sample
Normalisation - attempt to make the data look like everybody else’s data.
Differential expression analysis- difference of expression between our test and control samples. 
Biological interpretation- clustering analysis, geneset enrichment, pathway or network analysis.
Submit data to a public repository.
What is clustering?
Increasing gene expression and increase in body mass index. Organises data with similar patterns into classes - we are looking to see if there are any patterns in the data.
Objects within a class are more similar to each
other than to objects outside the class
Can be displayed on a dendrogram.
What are Dendrograms?
Dendrograms – “Trees”
Alternative way of displaying
similarity between samples
Distant samples are less similar
Eg. In osteoartheritis, there is a whole group of genes there not expressed in the normal, if we compare them side by side. We can tell if someone has it at one glance.
Eg. This can also be done in identifying cancer.
What happens in qPCR?
Reverse transcriptase is used on RNA to form complementary DNA (cDNA) for PCR.
We would PCR the gene of interest. Also PCR housekeeping gene. This is a gene widely expressed in all cells and tissues.
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. 
How is the exponential amplification in PCR dealt with?
If you begin with one sample, you can end with 17 billion copies after the 35th cycle for example. Virus if you started 15 samples, you could end up with 257 billion copies of the 35th cycle.
To establish a point where we can compare, we pick a common point.
fluorescence above background at 225 copies=Ct (threshold cycle)
So the higher the amount of starting RNA, the lower the Ct value. Ie. We need less cycles to make 225 copies.
How do you count the number of amplified molecules present?
Include a dye in the PCR reaction makes that fluoresces when it burns double-stranded DNA.
Label a probe in the PCR that only fluoresces when it is incorporated in the PCR product.
Why do we use qPCR?
- 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
There would be one in the clinic for instance as EndoPredict Risk Estimation for Breast cancer. It tests for whether chemotherapy will be effective or not.
What is the Breast Cancer EndoPredict Risk Estimation?
- 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.
qPCR tests would be used in the clinic.
In EndoPredict tests for 12 genes activity.
How do SNPs work in 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
