Microarrays Flashcards

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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)
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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
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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)
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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
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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
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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)
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7
Q

Commercial Microarray Instruments

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