Microarrays Flashcards
What is a microarray?
An ordered assembly of nucleic acids immobilised on a solid support
What is usually the solid support in microarrays?
The solid support is usually glass
What is transcriptomics and what does it allow?
Study of transcription and their functions
Allows to classify samples
What is there in microarrays for gene expression?
Lots of copies of the same probe in a spot
What does each spot in microarrays for gene expression give?
Each spot gives the relative expression for one transcript
What do microarrays for gene expression detect?
Detect all known transcripts in one sample
When we scan the solid support in microarrays, what do we end up with?
We end up with red, green and yellow spots
What does each spot represent?
Represents one SNP
Whats involved in expression profiling workflow?
-Sample–>RNA extraction–>Labelling–>Hybridisation of labelled sample to array–>Detect a signal–>Data analysis
What needs to be done once you have collected raw data?
Microdata analysis
Steps involved in microdata analysis
Normilisation–>Hierarchial clustering–>Gene filtering–>Statistical test–>Generate gene list–>Biological interpretation
What is clustering?
A technique that organises data with similar patterns into classes
What is an alternative way of displaying similarities between samples?
Alternative ways are dendrograms
-Distant samples are less similar
What is also used in clustering?
Heat maps
Why do we use data repositories?
- Microarray experiments aren’t cheap, so this maximises utility
- Can share data
- Easier to compare results
Techniques to predict cancer recurrence
Mamma print: -Uses microarrays -70 genes are observed Oncotype DX: -qRT-PCR used -21 genes are observed Mammostrat: -Immunohistochemistry used -5 proteins observed Prosigna gene signature: -58 genes observed -Direct digital barcode used
What do can we do in RT-PCR?
-Do PCR on cDNA and look whether things are expressed or not
How can we make RT-PCR quantitative?
We can make it quantitative by counting the number of copies of amplified DNA present
What is the Ct value and what does it stand for?
Stands for threshold cycle
Is a value to compare across all experiments and is fluorescence at 225 copies of the transcript
Relation between cDNA and Ct value
The higher the amount of cDNA, the lower the Ct value
How can you count the number of amplified molecules present?
- Include a dye in PCR reaction mix that fluoresces when its incorporated in the PCR product
- Or label a probe in the PCR that only fluoresces when its incorporated in the PCR product
Why do we use qPCR?
- Used to independently confirm differences in RNA levels between samples
- Probe binding is noisy and differences can be detected that aren’t real, especially when differences are small
What is a more accurate measure of RNA transcript abundance?State an advantage and disadvantage
RNA-seq
- Data more reproducible
- But its expensive
Why is GWAS possible and how?
GWAS only possible because we can genotype large number of SNPs in large number of subjects
- Possible by using microarrays that hybridise with genomic DNA adjacent to SNPs
- SNP then extended by one base that is fluorescently labelled and detected using a HD scanner
What is a spot?
Its lots of copies of the same single stranded oligonucleotide
-A probe essentially
What is each probe for?
Each probe is for genotyping one SNP
What is each probe designed to do?
Designed to hybridise with one SNP
What are copy number variants?
Sequences which are greater than 1kb that have different copy numbers in different people
Range in size and can see repeats or absentees.
Structural variants of copy number variants
- Arise due to:
- Deletion
- Insertion
- Inversion
Steps involved in Array-CGM
- Patient DNA taken and labelled green with fluroesence and control labelled red
- Control and patient DNA are mixed and hybridised to array of DNA fragment
What do red and green areas show in array CGM?
Show difference between normal and patient DNA
