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
What does each probe bind to?
→ a SNP
Describe how a microarray to find SNPs works?
→ Probes are attached to the slide
→ Take the fragmented genomic DNA of the patient and wash it over the slide
→ It hybridizes to the complementary probe
→ the immobilized probe is extended by one base using ddNTPs with a fluorescent tage
→ a laser triggers fluorescence and a sensitive scanner records the results
What percentage of the genome is copy number variants?
→ 12%
What are CNVs defined as?
→ sequences greater than 1kb that have different copy numbers in different people
What are the 7 structural variants of genes?
→ Reference
→ Deletion
→ Insertion → Inversion → Tandem duplication → Dispersed duplication → Copy number variants
Describe how array comparative genomic hybridisation works?
→ Label patient DNA green
→ Control DNA is red
→Mix and hybridise them to the array
→ you expect each probe to be red and green - same proportion
→ occasionally you see red instead of yellow which means the copy number is different between the patient and the control
What are the 3 uses of microarrays?
→ Gene expression
→ SNP genotyping
→ Structural variant detection
What are array CGH good for?
Detecting deletions and duplications of genomes
What does the array-CGH profile show?
graph is usually at 0 as there are equal amounts of red and green signal.
Negative is a deletion.
