Microarrays

Question 1

What is a microarray?

Answer 1

→ An ordered assembly of nucleic acids immobilized on a solid support

Question 2

What is the support in a microarray?

Answer 2

→ Glass similar to a microscope slide

Question 3

What is transcriptomics?

Answer 3

→ Finding the level at which a gene is expressed in a sample

Question 4

Describe microarrays for gene expression?

Answer 4

→ 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

Question 5

What is the function of a microarray for gene expression?

Answer 5

→ Detects all known transcripts in one sample

Question 6

Describe expression profiling workflow?

Answer 6

→ Take the sample and extract RNA 
→ remove tRNA and rRNA
→ label with fluorescent tages
→ Hybridize them to the array 
→ Detect the signal

Question 7

What is normalisation and why is it done?

Answer 7

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

Question 8

What is clustering?

Answer 8

→ Organising data with similar patterns into classes
→ Objects within a class are more similar to each other than objects outside the class

Question 9

How do dendrograms work?

Answer 9

→ Distant samples are less similar

Question 10

Why do data repositories exist?

Answer 10

→ Microarray experiments aren’t cheap so it maximises utility

Question 11

What does reverse transcriptase do?

Answer 11

→ Converts RNA to cDNA

Question 12

What is the relationship between RNA and Ct value?

Answer 12

→ The higher the amount of starting RNA the lower the Ct value

Question 13

What is the Ct value?

Answer 13

→number of cycles required for the fluorescent signal to cross the threshold

Question 14

What is an intercalating dye?

Answer 14

→ It binds between the stacked DNA base pairs

Question 15

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

Answer 15

→ Include a dye that fluoresces when it binds double stranded DNA
→ Intercalating dyes

Question 16

What is another method of counting the amplified molecules?

Answer 16

→ Label a probe that only fluoresces when it is incorporated into the PCR product

Question 17

What is qPCR used for?

Answer 17

→ To independently confirm differences in RNA levels between samples

Question 18

What is an accurate measure of RNA transcript abundance?

Answer 18

→ RNA seq

Question 19

Why is qPCR used?

Answer 19

→ Probe binding is noisy

→ differences can be detected that are not real

Question 20

Why are GWAS studies possible?

Answer 20

→ You can genotype large numbers of SNPs in large numbers of subjects

Question 21

What kind of microarrays are done in GWAS?

Answer 21

→ Microarrays hybridize with genomic DNA adjacent to SNPs rather than RNA transcripts

Question 22

What is in a spot?

Answer 22

→ Lots of copies of the same single stranded oligonucleotide - a probe

Question 23

What is each probe for in a microarray?

Answer 23

→ Genotyping one SNP

Question 24

What is a probe?

Answer 24

→ A piece of ssDNA approx. 20-30 nucleotides long

Question 25

What does each probe bind to?

Answer 25

→ a SNP

Question 26

Describe how a microarray to find SNPs works?

Answer 26

→ 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

Question 27

What percentage of the genome is copy number variants?

Answer 27

→ 12%

Question 28

What are CNVs defined as?

Answer 28

→ sequences greater than 1kb that have different copy numbers in different people

Question 29

What are the 7 structural variants of genes?

Answer 29

→ Reference
→ Deletion
→ Insertion
→ Inversion
→ Tandem duplication
→ Dispersed duplication
→ Copy number variants

Question 30

Describe how array comparative genomic hybridisation works?

Answer 30

→ 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

Question 31

What are the 3 uses of microarrays?

Answer 31

→ Gene expression
→ SNP genotyping
→ Structural variant detection