20.02.07 Other arrays

Question 1

Array CGH

Answer 1

• Array comparative genomic hybridisation.

- Competitive hybridisation of a test sample and a control sample to identify gains or losses.

Question 2

BAC array

Answer 2

• Bacterial artificial chromosome array
• Large clones of 150-200kb
• Propagated in vectors in bacteria, purified and amplified. Spotted onto glass slides
• Resolution= 0.5-1Mb

Question 3

Pros of BAC array

Answer 3

• Due to their large size they are stable
• hybridisation specific (high signal to noise ratio)
• Accurate CNV calling
• Fewer uncertain significance CNVs detected

Question 4

Cons of BAC array

Answer 4

• Lower resolution
• Abnormalities could be missed in the gaps
• LOH/UPD not detected
• Expensive (dyes and fewer arrays per slide)
• Difficult to reproduce, so batch to batch variation.

Question 5

Oligonucleotide arrays

Answer 5

• Single stranded oligonucleotide fragments (25-85bp)
• Oligos are spotted onto glass slides or synthesised directly onto surface
• As oligos are small they can be packed more densely. Means more probes and higher resolution (50-200kb)
• Multiple samples can be run on a slide

Question 6

Advantages of oligonucleotide array

Answer 6

• Multiple patients can be run on a single slide (cheaper and more consistency)
• Reproducible construction (less batch to batch variation)
• Higher resolution (100bp)
• Multiple consecutive probes needed for a CNV call, so more accurate.
• More flexibility, higher density, customisation.

Question 7

Disadvantages of oligonucleotide array

Answer 7

• Low detection of mosaicism
• more aberrant probes needed to accurately identify CNVs.
• Poor signal to noise ratio, more false positives (due to small probe size)
• Some imbalances are too small to be verified (need MLPA)
• Cannot detect LOH or UPD
• CNVs of uncertain significance more frequent.

Question 8

What are expression arrays

Answer 8

-Comparison of gene expression of thousands of genes in two cells/tissues

Question 9

Steps in expression arrays

Answer 9

• Cellular RNA is reverse transcribed to form cDNA population
• cDNA is labelled (biotin, Cy3)
• Labelled cDNA is applied to array and allowed to hybridise. Quantitative.
• Comparison. Needs normalisation first to correct for background and inter-experimental variation

Question 10

Expression profiling in tumours

Answer 10

• Can identify up/down-regulated genes.
• delineate molecular subtypes (molecular signatures)
• Used to predict clinical outcome or response to a given treatment.
• Not yet accurate or reproducible enough to be used in diagnostic setting.

Question 11

What are microRNAs

Answer 11

Small noncoding RNAs, thought to have a regulatory role in cells

Question 12

Expression profiling of microRNAs

Answer 12

-Cancer cells show abnormal miRNA expression profiles (miRNAs downregulated in most tumours)

Question 13

Microarray based techniques for detecting epigenetic changes

Answer 13

• ChIP-chip (chromatin immunoprecipitation combined with microarray detection)
• DamID (DNA adenine methyltransferase identification)
• Bisulphite modification followed by microarray hybridisation

Question 14

How does ChIP chip work

Answer 14

• Chromatin immunoprecipitation combined with microarray detection
• Cells treated with cross linking agent (formaldehyde), proteins are covalently linked to DNA in situ.
• DNA/protein complexes are fragmented and precipitated using antibodies specific to proteins of interest.
• Identifies the at attached DNA fragment. Cross links are reversed and DNA is labelled with a fluorescent dye and hybridised to a genome wide microarray.

Question 15

Uses of ChIP chip

Answer 15

To identify protein binding sites, e.g. for transcription factors
-To identify promoter regions, enhancers repressors etc

Question 16

What is DamID

Answer 16

• DNA adenine methyltransferase identification
• Transcription factor or chromatin-binding proteins of interest are fused to DNA adenine methyltransferase
• Expressed in vivo
• Dam targets the binding site of its fusion partner.
• Dam methylates adenine in immediate vicinity.
• Methylated adenines are identified by purification or selective amplification, fluorescently labeleld and hybiridsed to genome0wide array.

Question 17

What are the advantages of DamID over ChIP chip

Answer 17

• DamID doesn’t require the use of antibodies

- Only handles DNA, avoiding complications with unstable RNA or proteins.

Question 18

What is bisulphite modification followed by microarray hybridisation

Answer 18

• CpG islands in promoters can by hypermethylated in cancer genomes.
• Sodium bisulphite converts cytosine to uracil but leaves 5-methylcytosine unchanged.
• Oligonucleotide probes on the array hybridise specifically to the converted or unconverted sequence.
• Promoter hypermethylation in p16 gene in CRC patients in Kashimiri population. Correlated with decreased expression of p16, leading to disease.

Question 19

RNAi cell micoarrays

Answer 19

• RNA interference
• Post transcriptional method of gene silencing.
• Used for loss of function studies.

Question 20

How to RNAi cell microarrays work

Answer 20

• RNAi reagents are printed on to glass microarray slides.
• Put into culture dishes
• Cells adhere to spots and internalise material so become transfected. Remaining cells form a non-stransfected lawn between spots
• Fixed and prepared for ummunofluorescence, staining for DNA, in situ hybridisation, apoptosis detection etc.