L15: Microarrays

Question 1

What is a microarray?

Answer 1

A substrate of some kind (e.g. a glass slide) on which we have thousands of copies of the same short stretches of DNA, named probes

Question 2

What are ‘probes’?

Answer 2

The same short stretches of DNA

Question 3

How are probes used in microarrays?

Answer 3

Probes are complimentary in sequence to things in sample, which we will have extracted the nucleic acid from that we want to interrogate

Question 4

What does the term ‘features’ to?

Answer 4

Features are spots where probes are arranged, which have thousands of copies of the same probe’s sequence (in that feature)

Question 5

What mechanism do microarrays employ?

Answer 5

They work by hybridisation (watson-crick base pairing between probe and sample)

Question 6

How do microarrays use hybridisation?

Answer 6

• DNA/ RNA sample we want to bind is called the Target
• Results are then read out by target being labelled with a fluorescent marker
• Fluorescence intensity of feature relates to amount of target DNA in sample
• Allows us to observe changes for specific genes

Question 7

Name the 3 main applications of microarrays

Answer 7

Gene expression, SNP arrays/ affymetrix SNP6 arrays & methylation arrays

Question 8

How were microarrays were used for gene expression?

Answer 8

• arrays run under different conditions, allowing for differential gene expression as reported fold-changes

Question 9

What does use of microarrays in investigating gene expression allow?

Answer 9

Allows us to see what genes are switched on by cell response in different conditions; these can be investigated or characterisation

Question 10

Explain the use of SNP arrays/ affymetrix SNP6 arrays?

Answer 10

Probes are designed for different alleles, ratio of intensity allows us to see if SNP is reference or alternative allele
- copy # of each SNP can also be inferred via computer, allowing detection of chromosomal (large-scale) or focal duplications/ losses

Question 11

How are SNP arrays/ affymetrix SNP6 arrays useful?

Answer 11

They allow for GWAS, where gene linkage is found between SNP markers and phenotypes
- requires large patient cohorts

Question 12

What does ‘SNP’ stand for?

Answer 12

Single nucleotide polymorphism (change)

Question 13

How are methylation arrays carried out?

Answer 13

Via illumina 450K or EPIC methylation arrays

Question 14

How are methylation arrays useful?

Answer 14

Allows the methylation state of DNA to be interrogated

• important in epigenetic regulation of chromatin structure and in cancer
• cytosine bases that occur before a guanine can be methylated

Question 15

What can methylation of promoter regions of genes lead to?

Answer 15

The gene being silenced while being demethylated causing the over-expression of genes

Question 16

What does bisulfate treatment cause?

Answer 16

The conversion of cytosine to uracil and then thymine but methylation of cytosine at CpG sites protects bases from this conversion

• probes exist for each C or T state of a CpG
• ratio intensity here (beta-value) allows us to detect methylation state of CpG

Question 17

Explain the process of Northern Blotting

Answer 17

1. Sample of tissue mashed up then RNA extracted using nucleic kit
2. Then run on agarose gel (electrophoresis) with formaldehyde and seperated by size
3. Blot RNA onto nylon membrane since RNA probes can’t enter gel, creating complimentary bits of RNA identical to gene sequences interested in quantifying
4. Probes labeleed with fluorescent P and hybridise into this membrane and base pair against genes of interst
   Nylon membrane then put through beta decay and on x-ray film and placed in freezer or via phosphoimager freezer then developed

Question 18

Explain the process of microarrays

Answer 18

1. RNA extracted from sample and converted using reverse transcriptase to cDNA as mRNA isn’t stable. cDNA lablled w fluorescent dye
2. cDNA sample hybridised into microarray. Array has ssDNA probes in features and the more mRNA, the more fluorescence visible
3. Data is processed on computer and we end up with a fold change, due to comparing control to treated sample

Question 19

Why are microarrays now used over northern blotting? (nb)

Answer 19

• Microarrays are less time consuming and laborious as it’s an automated process
• Not radioactive, unlike nb
• Microarrays can work on 10,000 of genes, while nb can only work on a handful
• Microarrays provide much higher dynamic range but nbs provides mainly qualitative data
• Microarrays don’t need hypothesis, unlike nb, in advance to decide which genes wanted to be investigated

Question 20

How do spotted cDNA microarrays work?

Answer 20

Spot cDNA probes onto microscopic slides and later robots used to spot slides
- common to run 2 samples on the same arrray using green Cy3 and red Cy5, as one channel would be control

Question 21

What have spotted cDNA microarrays been replaced with?

Answer 21

Commerically produced arrays, which use oglionucleotide prones synthesised on a chip in place of bound cDNAs on glass slide

Question 22

Why are commercially produced arrays preferred over spotted cDNA microarrays?

Answer 22

• commercial arrays have probes synthesised in situ on chip
• results are more comparable due to standard experimenal procedures, automation for sample prep and hybridisation and washing
• no complicated dye swapping experimental design
• uses cRNA targets labelled with single fluorochrome
• a single sample is hybridisd to a single chip

Question 23

Name limitations of microarrays

Answer 23

It’s not easy to compare them due to

• difference in sample extraction yield (mRNA)
• differences in hybridisation conditions
• differences from one array to the next and possible artefacts on array

Question 24

How is quality control ensured on microarrays?

Answer 24

• Have 3 of each array, minimum
• Batch effect: show up where sample variability is due to sampling handle, rather than biological variation e.g. were samples extracted/ hybridised on the same day or by the same person

Question 25

Why is replication os samples required for statistical analysis?

Answer 25

Array data is noisy and easily pertubed

Question 26

Why are proteomics used?

Answer 26

Detecting mRNA level may not be indicative of increased protein production