Microarrays Flashcards
What is a microarray?
An ordered assembly of nucleic acids immobilized on a solid support
What is the support in a microarray?
Glass similar to a microscope slide
What is transcriptomics?
Finding the level at which a gene is expressed in a sample
Describe microarrays for gene expression
→ Lots of copies of the same probe in a spot
→ Each spot gives the relative expression for one transcript
→ Each spot represents one SNP
→ They allow us to analyse genetic markers across the genome
What is the function of a microarray for gene expression?
Detects all known transcripts in one sample
Describe expression profiling workflow?
→ Take the sample and extract RNA
→ Remove tRNA and rRNA
→ Label with fluorescent tages
→ Hybridize them to the array
→ Detect the signal
What is normalisation and why is it done?
Making sure that there aren’t any samples that bind preferentially for reasons other than the fact that they are expressed
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
How do dendrograms work?
Distant samples are less similar
Why do data repositories exist?
Microarray experiments aren’t cheap so it maximises utility
What does reverse transcriptase do?
Converts RNA to cDNA
What is the relationship between RNA and Ct value?
The higher the amount of starting RNA the lower the Ct value
What is the Ct value?
Number of cycles required for the fluorescent signal to cross the threshold
What is an intercalating dye?
It binds between the stacked DNA base pairs
How do you count the number of amplified molecules present in PCR?
→ Include a dye that fluoresces when it binds double stranded DNA
→ Intercalating dyes
What is another method of counting the amplified molecules?
Label a probe that only fluoresces when it is incorporated into the PCR product
What is qPCR used for?
To independently confirm differences in RNA levels between samples
What is an accurate measure of RNA transcript abundance?
RNA seq
Why is qPCR used?
→ Probe binding is noisy
→ Differences can be detected that are not real
Why are GWAS studies possible?
You can genotype large numbers of SNPs in large numbers of subjects
What kind of microarrays are done in GWAS?
Microarrays hybridize with genomic DNA adjacent to SNPs rather than RNA transcript
What is in a spot?
Lots of copies of the same single stranded oligonucleotide - a probe
What is each probe for in a microarray?
Genotyping one SNP
What is a probe?
A piece of ssDNA approx. 20-30 nucleotides long
