Microarrays Flashcards
1
Q
Micorarray (Gene Chip) Technology
A
- 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)
2
Q
Comparison with Standard Blotting
A
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
3
Q
Concept of Microarrays - Probes
A
- 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)
4
Q
Concept of Microarrays - Spots
A
- 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
5
Q
Concept of Microarrays – Scope
A
- 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
6
Q
Microarray Preparation
A
- 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)
7
Q
Commercial Microarray Instruments
A
- NIEHS microarrayer
(Beecher Instruments, Inc.) - Affymetrix gene chips
8
Q
GeneChip analysis process
A
- 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
9
Q
Oligonucleotide Arrays - Preparation
A
- 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
10
Q
Photolithography
A
- 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”
11
Q
Gene Chip (Affymetrix)
A
- 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
12
Q
Hybridisation Procedures
A
- 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)
13
Q
A typical Gene Expression Assay
A
- Competitive binding.
- Colour spectrum // relative amounts of A vs B
- control (Cy3) + treated sample (Cy5)
- equal expression
14
Q
Advantages of microarrays
A
- 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
15
Q
Disadvantages of microarrays
A
- 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