Microarrays

Question 1

Micorarray (Gene Chip) Technology

Answer 1

• Multiplex “assay”
• Array of thousands of DNA probes per experiment
• DNA can be parts of genes, cDNAs etc. with known sequence
• Can screen either: many samples for several DNA sequences, few samples for many DNA sequences
• Similar to Southern blotting but DNA spots bound covalently to solid surface (glass or silicon)

Question 2

Comparison with Standard Blotting

Answer 2

Southern or Northern Blot
- labelled, identical
probe molecules
- immobilised target DNA or RNA (from tissues/cells)
- nylon membrane
Gene chip
- different, immobilised
probe molecules
- labelled target molecules
e.g. RNA from cells
- glass support

Question 3

Concept of Microarrays - Probes

Answer 3

• Probes spotted (cDNA) or built (oligonucleotide) onto a non-porous gridded glass support (slide)
• 10^6 - 10^9 identical copies per spot
• cDNA probes: 100s nucleotides in length (e.g. specific to a complete gene)
• Oligo probes 20-25 nucleotides in length (e.g. specific to part of gene)

Question 4

Concept of Microarrays - Spots

Answer 4

• Spot volumes: 0.25-1.0 nL
• Spot diameter: ~100 mm
• Spacing between spot centres: ~250 mm
• Many spots (~6000) will fit onto glass slide (e.g. 60×100 spots can fit on a 1.485 cm × 2.485 cm = 3.69 cm2 surface area)
• Spots are air-dried & DNA is covalently-linked to glass surface by UV treatment

Question 5

Concept of Microarrays – Scope

Answer 5

• An array of different cDNAs or oligonucleotides allows 1000s of hybridisation tests simultaneously
• Analysis of expression levels of 1000s mRNA molecules in parallel (transcriptomics): developmental studies, drug-testing, cancer research etc.
• DNA variation screening: mutation detection, SNPs etc., relies on stringency

Question 6

Microarray Preparation

Answer 6

• Semi-manually (cDNAs) or purchased from commercial suppliers (cDNA & oligo arrays)
• Robotic pipetting essential for accurate X-Y positioning and consistent spot dimensions
• Inkjet technology sometimes used - especially in commercial equipment
• Liquid “pens” must give reproducible & precise spots
• Stainless steel microarray spotting tips
• <10-500 nL sample taken from 96, 384, 1536, etc..-well plate for spotting
• Acoustic spotting methods (2.5 nL)

Question 7

Commercial Microarray Instruments

Answer 7

• NIEHS microarrayer
  (Beecher Instruments, Inc.)
• Affymetrix gene chips

Question 8

GeneChip analysis process

Answer 8

• GeneChip p53 probe array
• each probe cell contains millions of copies of a specific oligonucleotide probe
• fluorescein-labelled DNA target from experimental sample added
• image of hybridized probe array

Question 9

Oligonucleotide Arrays - Preparation

Answer 9

• Commercially-prepared: base by base in situ synthesis using photolithography
• Nucleotides protected with photosensitive group
• Photoactivation (light) removes protecting group
• Nucleotide now able to react with next nucleotide in sequence
• Masks used to permit activation of specific spots only

Question 10

Photolithography

Answer 10

• light passes through a photolithographic mask onto glass surface (at specific points only)
• one mask for each specific base for each nucleotide in the sequence
• based on early computer chip creation - “DNA chip”

Question 11

Gene Chip (Affymetrix)

Answer 11

• 1.28 x 1.28 cm (varies)
• 280,000 different probes
• Plus replicates included (>2x)
• each feature interrogated by >20 pairs of oligos
• ~10^3 - 10^5 genes / transcripts / splice variants

Question 12

Hybridisation Procedures

Answer 12

• Similar to high-stringency Southern or northern blotting
• Probes are unlabelled and fixed to glass substrate
• Sample DNA and Control DNA labelled with different fluorophore
• Automated hybridisation, washes, image scanning
• Software analyses data (SNR) to produce relative expression levels: internal standard (e.g. actin, GAPDH), external standard (e.g. control sample: reference)

Question 13

A typical Gene Expression Assay

Answer 13

• Competitive binding.
• Colour spectrum // relative amounts of A vs B
• control (Cy3) + treated sample (Cy5)
• equal expression

Question 14

Advantages of microarrays

Answer 14

• High-densities of probes per chip
• Automation – rapid
• Multiple samples per assay
• Increased sensitivity, specificity and dynamic range
• Massive amounts of data from small sample

Question 15

Disadvantages of microarrays

Answer 15

• Commercial product: Affymetrix Inc.
• Machines + software ~£125,000
• Single use chips
• False results, operator error, and day-to-day variance
• Influenced by: RNA secondary structure: higher affinity probes (e.g. 2’-OMe, LNA), probe crowding & base composition
• Requires complex & careful statistical analysis