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 transcript

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 tag

→ 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

Question 32

What are array CGH good for?

Answer 32

Detecting deletions and duplications of genomes

Question 33

What does the array-CGH profile show?

Answer 33

Graph is usually at 0 as there are equal amounts of red and green signal. Negative is a deletion.