Microarrays 1 Flashcards
What are microarrays?
Ordered assembly of nucleic acids immobilised onto a solid support.
(essentially thousands of probes attached onto the solid surface)
- DNA samples are complementary to the oligonucleotide sequence probes on the slide and will hybridise
- This can then be detected by fluorescence
What is transcriptomics?
The study of all the RNA molecules within a cell
- Can use it to look at expression levels of genes in samples, see if genes are up/down regulated in diseased cells
- Discover biology of samples, classify them and see which class a sample belongs to
How are microarrays used to measure gene expression?
- There are spots which contain lots of copies of the same probe (ssDNA isolated onto the array)
- Each spot gives the relative expression for one transcript
- This allows you to detect all the known transcript in one sample
- It does this by changing the colour of the spot if this sample is present or not
How is profiling workflow carried out?
- Two samples test and control
- Convert the mRNA to cDNA and labelled with a dye (different colour depending on the sample)
- cDNA samples are mixed and hybridised onto the array
- The chip (microarray) is then scab with a laser to detect the signal
- You will then perform data analysis
- e.g If the gene is expressed in the test sample it will be red, if the sample is expressed in the control it will be green, if it is expressed in both it will be yellow
What do you do after you obtain the raw data from the microarray?
1) NORMALISATION = make sure there is no other reason why the probes are binding to the sample other than the fact they’re being expressed
2) HIERARCHAL CLUSTERING
3) GENE FILTERING (filter out the genes you arent interested in)
4) STATISTICAL TESTS: perform tests to determine significance
5) GENERATE GENE LIST: create a list of interesting findings using pathways analysis (take all the genes and show which ones are up/down regulated or dont change in our disease or biological trait
6) BIOLOGICAL INTERPRETATION - impose biological interpretation on results
Explain one of the methods of gene expression analysis?
Hierarchal clustering is one of the methods of gene expression analysis
- This organises data with similar patterns into classes, objects within a class are more similar then objects outside of the class
- e.g some genes expressed at a low BMI and others at a high
- DENDOGRAMS = is another way of clustering, distant samples are less similar and visa versa
- pink and green dots are more similar in terms of gene expression
How did data repositories come about?
- Microarray experiments aren’t cheap, so to maximise utility we share the data and use other peoples 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 is
- ArrayExpress
- EBI (European Bioinformatics Institute)
- GEO (Gene Expression Omnibus)
- NCBI (National Centre for Biotechnology Information)
- An example is
How are microarrays used in the NHS?
- The NHS uses microarrays for breast cancer management and chemotherapy decisions
- Some gene expression profiles and expanded immunohistochemistry tests will be carried out
- Tests like this ensure that only patients which benefit from chemotherapy will recieve it
- For example Breast Cancer EndoPredict Risk Estimation
- Tests like this ensure that only patients which benefit from chemotherapy will recieve it
How does RT-PCR link with microarrays?
- qPCR is used to convert microarray results
- RNA will be converted to cDNA using reverse transcriptase
- This is then amplified using PCR and run on a gel
- The sample is then analysed and compared using a housekeeping gene
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This is carried out to see if certain elements are being expressed or not
- e.g in the diagram the differences in the gel above should be due to experimental variation in the amount of RNA and be corrected
How can you make RT-PCR quantitative?
By counting the number of copies of amplified DNA present using flourescent molecules ‘tags’
What is a Ct value in qPCR and what does it mean?
Ct value = Fluorescence above background at 225 copies
The higher the amount of starting RNA (cDNA) the lower the Ct value
- This is because you will reach the fluorescence above background at 225 copies a lot quicker
- e.g only 5 cycles are required when the number of copies is 5 compared to when the number of copies is 1
Why is qPCR used?
- qPCR is used to independantly confirm differences in RNA levels between samples
- Probe binding is noisy and differences can be detected that are not real where the differences are small
- 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
How do you count the number of molecules present in RT-PCR?
- 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
How do SNP microarrays happen?
- GWAS (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 which hybridise genomic DNA adjacent to SNPs (rather than RNA transcripts)
- The immobilised piece of DNA on the microarray is complementary to the DNA before the SNP (single bp before)
- The SNP is then extended by one base which is fluorescently labelled and detected using a high definition scanner
Whats in a spot?
- Lots of copies of the same single-stranded oligonucleotide - a ‘probe’
- Each probe is for genotyping one SNP
How do SNPs microarrays work?
- We take a probe and attach multiple copies to the same spot on a slide
- There will be multiple spots which have different probe DNA sequences depending on the SNP you are looking for
- The fragmented DNA is taken from the patient and washed over the slide which will hybridise to the complementary probe
- In this situation the patient will be heterozygous for the first SNP and homozygous for the second
- The immobilised oligonucleotide probe will be extended by one base using dideoxynucleotide triphosphates (ddNTPS) with a fluorescent tag
- A laser will trigger fluorescence and a scanner will record the results
- The patient is homozygous for the C allele however for this SNP, half the patient DNA contains a guanine at this position and half contains adenine meaning that the patient is heterozygous for this SNP
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How does array comparative genomic hybridisation work?
Used to analyse CNVs or structural variants in a test sample compared to a reference sample
- Patient DNA is labelled in green and control is labelled in red and mixed together
- These are hybridised onto an array which has probes with all the different regions in the genome
- You expect the colours to show in the same proportion as they were added = 1:1
- However may get results which are red when it should be yellow which suggests the copy number is different between the patient and control
- Suggets there is a duplication/deletion in a region that is important and related to the disease
