Microarrays

Question 1

What are microarrays?

What is a probe?

Answer 1

• A microarray is an ordered assembly of nucleic acids immobilised on a solid support o The support is usually glass
  ▪ Similar to a microscope slide
• The nucleic acid that is typically mobilised is DNA
• Short DNA sequences usually

Probes are the short pieces of single-stranded DNA immobilised on the surface of the array. They are oligonucleotides. Each spot on the array consists of thousands of probes with the same sequence

Question 2

Describe what can be seen on the diagram of a microarray?

Answer 2

On the left there is a zoomed in view of a microarray o There are known locations on the slide where we have arrayed lots of nucleotide strings.

There are millions of them and they are single stranded

Question 3

• What are the expression levels of all the genes in my samples?
• Which genes are expressed at different levels between two different types of samples?

Answer 3

o Discover the biology of your samples
o Classify samples
o Predict which class a sample belongs to

Question 4

Describe the arrangement of the microarrays

Answer 4

• Lots of copies of the same probe in a spot
• Each spot gives the relative expression for one transcript
• Detects all known transcripts in one sample

Question 5

What happens when we scan the slide?

Answer 5

• When we scan the slide we’d end up with an image containing red, green and yellow spots. - Each spot represents one SNP
• Because we have lots of spots we can analyse lots of spots simultaneously
• And microarrays therefore allow us to analyse genetic markers across the genome

Question 6

What occurs in Expression Profiling Workflow

Answer 6

• Take the sample and extract the RNA
• Purify it
• Label the sample with fluorescent tags
• Hybridise them
• Detect the signal
• And then do a lot of analysis e.g data analysis, including pathway analysis and biological interpretation

Question 7

What is microarray normalisation?

What is Hierarchical Clustering?

Give some examples of the data analysis that can occur in microarrays

Answer 7

• Normalisation o Making sure that there aren’t areas or probes that are particularly binding preferentially other than the fact that they are being expressed at that level
• Hierarchical Clustering - In hierarchical clustering, genes with similar expression patterns are grouped together and are connected by a series of branches (clustering tree or dendrogram).
• Gene Filtering
• Statistical Tests
• Generate Gene list
• Biological Interpretation

Question 8

What does clustering achieve?

Answer 8

• Organises data with similar patterns into classes

- Objects within a class are more similar to each other than to objects outside the class

Question 9

What is a dendrogram?

Answer 9

• Look like trees
• It’s an alternative way of displaying similarity between samples
• Distant samples are less similar

Question 10

What are heat maps?

Answer 10

In heat maps the data is displayed in a grid where each row represents a gene and each column represents a sample. The colour and intensity of the boxes is used to represent changes (not absolute values) of gene expression.

• Clustering is based on the expression of the gene of interest
• The red ones are downregulated
• Heat maps can be used diagnostically

Question 11

Microarrays are very expensive, how can we keep costs down?

Answer 11

Microarray experiments aren’t cheap, so to maximise utility we can:
share data and use other people’s data

Question 12

When comparing results what makes this process easier?

Who can provide these comparisons?

Answer 12

• If users provide the Minimum Information About a Microarray Experiment (MIAME) then it is easier to compare results
• ArrayExpress, EBI (European Bioinformatics Institute)
• GEO – Gene Expression Omnibus, NCBI (National Center for Biotechnology Information, USA)

Question 13

What does a gene expression pattern show, using the tumour as an example?

Answer 13

• Looking left to right, each row represents a tumour
• Top to bottom, they are different probes
• You can see that there are differences between group A and B o Can clearly see that there is more than one type of tumour

Question 14

What methods can be used to predict for cancer recoccurance?

Answer 14

On table

Question 15

How can we make RT-PCR quantitative?

What is the Ct value?

Answer 15

• DNA to RNA to Protein
• However reverse transcriptase can convert RNA to cDNA o we can do PCR on the cDNA and can look whether things are expressed or not

Question 16

How can we make RT-PCR quantitative?

Answer 16

• We can make RT-PCR quantitative by counting the number of copies of amplified DNA present o We count the copies by using fluorescent molecules
  ▪ Tags

Question 17

What is Ct?

Answer 17

Ct value is an arbitrary value that allows you to compare across all experiments o It’s the fluorescence at 225 copies of the transcript
▪ In the first sample, its after 9 cycles
▪ In the second sample, its after 5 cycles

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

Question 18

How do you count the number of amplified molecules present?

Answer 18

• 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
• Intercalating dyes are so-called because they bind between the stacked DNA base pairs

OR

• Label a probe in the PCR that only fluoresces when it is incorporated in the PCR product,
e.g. TaqMan (essentially labelling the PCR primer)

Question 19

Have a look at a DNA Amplification Plot for Q-PCR

Answer 19

On document

Question 20

Why use qPCR?

Answer 20

• qPCR is 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 (<2-fold)
• RNA-Seq is a more accurate measure of RNA transcript abundance, it is more reproducible and works over a wider range of concentrations…..but it is more expensive

Question 21

Why are GWAS’ possible?

Answer 21

• Genome-wide Association Studies are only possible because we can genotype large numbers of SNPs in large numbers of subjects
• This is possible 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 22

What is found in each spot?

Answer 22

• Lots of copies of the same single-stranded oligonucleotide - A “probe”
• Each probe is for genotyping one SNP
• Each spot contains lots of copies of the same oligonucleotide probe.
• This is a single stranded piece of DNA approximately 20-30 nucleotides long.
• Each probe is designed to hybridise with one SNP.

Question 23

What do we then do with this probe?

Answer 23

• We then take that probe and attach it to a glass slide.
• But not just one copy, lots of copies of the same probe, all being stuck in a spot on the slide.
• And we do the same thing for the next SNP we’re interested in - And we do that for all the SNPs we’re interested in.
• We then take fragmented genomic DNA from our patient and wash it over the slide - It will then hybridise, to its complementary probe.
• You can see in this situation the patient is heterozygous for the first SNP and homozygous for the second
• The immobilised oligonucleotide probe is extended by one base using dideoxy nucleotide triphosphates (ddNTPS) with a fluorescent tag.
• A laser then triggers fluorescence and a very sensitive scanner records the results.
• Software then transforms this into a genotype
• The C is detected by a red tag, the T by a green tag and the G by a red tag too

Question 24

How can microarrays be used for genotyping?

Answer 24

• There are lots of copies of the same probe in a spot
• Each spot gives the genotype for one SNP
• Up to 5 million spots per sample
• Genome wide analysis is possible
  ➢ When we scan the slide we’d end up with an image like this, containing red, green and yellow spots.
  ➢ Each spot represents one SNP
  ➢ Because we have lots of spots we can analyse lots of spots simultaneously
  ➢ And microarrays therefore allow us to analyse genetic markers across the genome

Question 25

What is Array CGH?

Answer 25

Array CGH is a technique which screens the whole genome to detect copy number changes (unbalanced gains/duplications and losses/deletions of genetic material) which may be contributing to a child's phenotype. Copy number variants - ~12% of the genome = CNV - Typically defined as sequences greater than 1kb that have different copy numbers in different people - >2000 have been identified o 10kb-5000kb o Can be repeated/absent

Question 26

Have a look at Structural Variants and Copy Number Variants

Answer 26

On document

Question 27

What occurs in Array Comparative Genomic Hybridisation (aCGH)?

Answer 27

On image - Green dye is Cy3, red dye is Cy5 - The output of the scanning process is the log of the ratio of the fluorescence intensities for each spot – usually this is 0 as there are equal amounts of red and green signal.