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πšπšŽπš—πš˜πš–πš’πšŒπšœ > Microarrays > Flashcards

Microarrays Flashcards

1
Q

What is a microarray?

A

β†’ An ordered assembly of nucleic acids immobilized on a solid support

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2
Q

What is the support in a microarray?

A

β†’ Glass similar to a microscope slide

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3
Q

What is transcriptomics?

A

β†’ Finding the level at which a gene is expressed in a sample

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4
Q

Describe microarrays for gene expression?

A

β†’ Lots of copies of the same probe in a spot
β†’ Each spot gives the relative expression for one transcript
β†’ Each spot represents one SNP
β†’ They allow us to analyse genetic markers across the genome

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5
Q

What is the function of a microarray for gene expression?

A

β†’ Detects all known transcripts in one sample

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6
Q

Describe expression profiling workflow?

A 
β†’ Take the sample and extract RNA 
β†’ remove tRNA and rRNA
β†’ label with fluorescent tages
β†’ Hybridize them to the array 
β†’ Detect the signal
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7
Q

What is normalisation and why is it done?

A

β†’ Making sure that there aren’t any samples that bind preferentially for reasons other than the fact that they are expressed

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8
Q

What is clustering?

A 
β†’ Organising data with similar patterns into classes
β†’ Objects within a class are more similar to each other than objects outside the class
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9
Q

How do dendrograms work?

A

β†’ Distant samples are less similar

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10
Q

Why do data repositories exist?

A

β†’ Microarray experiments aren’t cheap so it maximises utility

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11
Q

What does reverse transcriptase do?

A

β†’ Converts RNA to cDNA

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12
Q

What is the relationship between RNA and Ct value?

A

β†’ The higher the amount of starting RNA the lower the Ct value

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13
Q

What is the Ct value?

A

β†’number of cycles required for the fluorescent signal to cross the threshold

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14
Q

What is an intercalating dye?

A

β†’ It binds between the stacked DNA base pairs

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15
Q

How do you count the number of amplified molecules present in PCR?

A

β†’ Include a dye that fluoresces when it binds double stranded DNA
β†’ Intercalating dyes

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16
Q

What is another method of counting the amplified molecules?

A

β†’ Label a probe that only fluoresces when it is incorporated into the PCR product

17
Q

What is qPCR used for?

A

β†’ To independently confirm differences in RNA levels between samples

18
Q

What is an accurate measure of RNA transcript abundance?

A

β†’ RNA seq

19
Q

Why is qPCR used?

A

β†’ Probe binding is noisy

β†’ differences can be detected that are not real

20
Q

Why are GWAS studies possible?

A

β†’ You can genotype large numbers of SNPs in large numbers of subjects

21
Q

What kind of microarrays are done in GWAS?

A

β†’ Microarrays hybridize with genomic DNA adjacent to SNPs rather than RNA transcripts

22
Q

What is in a spot?

A

β†’ Lots of copies of the same single stranded oligonucleotide - a probe

23
Q

What is each probe for in a microarray?

A

β†’ Genotyping one SNP

24
Q

What is a probe?

A

β†’ A piece of ssDNA approx. 20-30 nucleotides long

25
Q

What does each probe bind to?

A

β†’ a SNP

26
Q

Describe how a microarray to find SNPs works?

A

β†’ Probes are attached to the slide
β†’ Take the fragmented genomic DNA of the patient and wash it over the slide
β†’ It hybridizes to the complementary probe
β†’ the immobilized probe is extended by one base using ddNTPs with a fluorescent tage
β†’ a laser triggers fluorescence and a sensitive scanner records the results

27
Q

What percentage of the genome is copy number variants?

A

β†’ 12%

28
Q

What are CNVs defined as?

A

β†’ sequences greater than 1kb that have different copy numbers in different people

29
Q

What are the 7 structural variants of genes?

A 
β†’ Reference
β†’ Deletion
β†’ Insertion
β†’ Inversion
β†’ Tandem duplication
β†’ Dispersed duplication
β†’ Copy number variants
30
Q

Describe how array comparative genomic hybridisation works?

A

β†’ Label patient DNA green
β†’ Control DNA is red
β†’Mix and hybridise them to the array
β†’ you expect each probe to be red and green - same proportion
β†’ occasionally you see red instead of yellow which means the copy number is different between the patient and the control

31
Q

What are the 3 uses of microarrays?

A

β†’ Gene expression
β†’ SNP genotyping
β†’ Structural variant detection

πšπšŽπš—πš˜πš–πš’πšŒπšœ (19 decks)

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