Microarrays

Question 1

What is a Microarray?

Answer 1

An ordered assembly of nucleic acids immobilised on a solid support.

Question 2

What support is used in a microarray?

Answer 2

Glass or something similar to a microscope slide

Question 3

Describe a simple microarray technique

Answer 3

1. An illumina Omni5 microarray for SNP genotyping - there are five million different immobilised oligonucleotides in each section that starts with the short double block and so this array can be used to genotype four samples.
2. A view of a small part of the array through a microscope so that the spots are visible.
3. A single-stranded DNA molecule hybridised to its target DNA in one of the spots connected to the solid support with the buffer solution around it.

Question 4

Explain how DNA is hybridised using the microarrays method

Answer 4

• Single-stranded oligonucleotides immobilised on the array so that target DNA or RNA can hybridise to it.
• After the DNA is hybridised, it is labelled, shine a laser and use a scanner to pick up the hybridised DNA.

Question 5

Explain the application of microarrays

Answer 5

How many genes are expressed in a particular tissue or cancer at a specific time - use a gene expression array to see the whole set of RNA expressed at a specific time

Question 6

What is transcriptomics?

Answer 6

The study of the transcriptome - the complete set of RNA transcripts that are produced by the genome.

Question 7

How does microarrays allow us to analyse genetic markers across the genome?

Answer 7

• Lots of the copies of the same probe - single stranded DNA isolated in a spot.
• Scan the slide and it contains red, green and yellow spots.
• Each spot gives the relative expression for one transcript so detects all known transcripts in one sample.
• Lots of spots can be analysed simultaneously.
• Therefore allowing us to analyse genetic markers across the genome.

Question 8

Which type of colour array is used more commonly?

Answer 8

Two colour array - the experiment sample in one colour and the control sample in another.

Question 9

What is the data analysis workflow and why is it used?

Answer 9

Feature extraction -> Quality control -> Normalisation -> Differential Expression analysis -> Biological interpretation -> Submit data to a public repository

It is used to find out how much RNA is there and then expression analysis.

Question 10

What is one way to do expression analysis?

Answer 10

Hierarchical dressing/clustering

Question 11

What is clustering?

Answer 11

• Organises data with similar patterns into classes
• Objects within a class are more similar to each other than to objects outside the class
• The genes which are closert together, the more likely they are to be part of the same mechanism. The further away, the less likely.

Question 12

What are dendrograms?

Answer 12

“Trees” - an alternative way of displaying similarity between samples

Question 13

When samples are more distant what does this mean?

Answer 13

Distant samples are less similar - in terms of their gene expression NOT sequence

Question 14

What is a heat map?

Answer 14

Taken out the vast majority of genes and left the ones that refer to normal genes and one specific disease

Question 15

Why are data repositories used?

Answer 15

Because microarray experiments aren’t cheap, so maximise utility and share other data, use other people’s data.

Question 16

What is the MIAME?

Answer 16

It is the minimum information about a microarray experiment (MIAME) then it is easier to compare results.

Question 17

How do we confirm microarray results?

Answer 17

Using a different technique needs to be used to confirm microarray results - Quantitative PCR (QPCR)

Question 18

What is the central dogma?

Answer 18

DNA -> RNA -> Protein

Question 19

What is reverse transcriptase?

Answer 19

It is an enzyme used by retroviruses to convert their RNA genomes into DNA.

Question 20

Why is reverse transcriptase used?

Answer 20

• It is used in the lab to convert the RNA into cDNA (complementary DNA).
• Perform PCR on cDNA and run the products out on a gel - RNA is not robust enough.

Question 21

What do bands represent on a gel product?

Answer 21

Some bands will be stronger than others, this means that the gene is more highly expressed in that tissue.
Fainter band suggests a lower level of expression (kidney and liver).

Question 22

What is a housekeeping gene?

Answer 22

Gene expressed in all tissues at a similar level (e.g. GAPDH, beta-actin)

Question 23

Why are housekeeping genes used?

Answer 23

Housekeeping genes are expressed at the same level in all tissues and there may be strong band in all samples, to suggest that we started with the same amount of RNA in all samples.

Question 24

Why else is RT-PCR used?

Answer 24

To look at the length of the transcript produced.

Question 25

How can a RT-PCR be made quantitative?

Answer 25

By counting the number of copies of amplified DNA present.

Question 26

How are copies of amplified DNA counted?

Answer 26

It is counted by using fluorescent molecules - "tags".

Question 27

What is the Ct value?

Answer 27

It is the threshold cycle - fluorescene above background at 225 copies.

Question 28

Describe the relationship between Ct and amount of RNA

Answer 28

The higher the amount of starting RNA (cDNA), the lower the Ct value (less cycles).

Question 29

How do you count the number of amplified molecules present?

Answer 29

- Include a dye in the PCR reaction mix that fluoresces when it binds double-stranded DNA e.g. an intercalating dye such as SYBR Green. - Label a probe in the PCR that only fluoresces when it is incorporated in the PCR product e.g. TaqMan

Question 30

What is another name for RT-PCR and should be used more?

Answer 30

qPCR is the same as RT-PCR. It is now considered best practice to describe PCR amplification and quantitation of the product.

Question 31

Why is qPCR used?

Answer 31

- Used to independently confirm differences in RNA levels between samples. - Probe binding is noisy, and differences can be detected that are not real, especially where differences are small. - RNA-Seq is a more accurate measure of RNA transcript abundance, it is more reproducible and works over a much wider range of concentrations but it is more expensive.

Question 32

How is qPCR information used in the NHS?

Answer 32

For tumour profiling tests for early breast cancer treatment decisions

Question 33

What is the EndoPredict Risk Estimation?

Answer 33

This test ensures that only patients who will benefit from chemotherapy receive it. - At low score (EPclin <3), endocrine therapy (ET) alone is sufficient. - At higher scores, ET + C is clearly beneficial compared to ET alone.

Question 34

Why are GWAS possible?

Answer 34

They are possible because we can genotype large numbers of SNPs in large numbers of subjects

Question 35

What do SNP microarrays do?

Answer 35

By using microarrays that hybridise with genomic DNA adjacent to SNPs (rather than RNA transcripts). The SNP is then extended by one base that is fluorescently labelled and detected using a high definition scanner.

Question 36

What is a spot on the microarray?

Answer 36

Contains lots of copies of the same oligonucleotide probe. This is a single stranded piece of DNA approximately 20-30 nucleotides long.

Question 37

How does an SNP microarray work?

Answer 37

1. Take the DNA sequence and design a probe complementary to the region next to the SNP 2. Take the probe and attach it to a glass slide 3. Add lots of copies of the same probe, all being stuck in a spot on the slide. 4. Do it for all the target SNPs 5. Take fragmented genomic DNA from our patient and wash it over the slide 6. It will then hybridise, to its complementary probe 7. The immobilised oligonucleotide probe is extended by one base using dideoxy nucleotide triphosphates with a fluorescent tag. 8. A laser triggers fluorescence and a very sensitive scanner records the results. The software transforms it into a genotype. 9. This will show the heterozygous and homozygous SNPs that the patient has. 10. Then a colour is added to these SNPs.- This makes the spot. Spots represent one SNP.

Question 38

What does software do to SNP genotypes?

Answer 38

- Software translates the three different colour signals for each probe into genotypes. - Use the cluster analysis to decide where the genotypes are - A few SNPs are reviewed by hand (<50) but most are not.

Question 39

What can an array profile be used for?

Answer 39

Used to find structural variants and copy number variants like duplications and deletions

Question 40

What detects structural variants?

Answer 40

Array CGH - however many of these applications have been replaced with Next-Gen sequencing protocols