Microarrays

• 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)

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

• 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)

• 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

• 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

• 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)

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

• 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

• 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

• 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”

• 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

• 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)

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

• 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

• 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