Laboratory techniques

The techniques described here are presented as an outline to the methodology; individual differences to the methods will be presented in subsequent chapters.

Quality assessment of DNA

The concentration and 230/240 and 260/280 ratios were measured using the Nanodrop® spectrophotometer ND-1000 (Thermo scientific, Delaware, USA). The 260-280nm ratio of samples needed to fall within 1.8 to 2 to be uncontaminated and be used in this study.

Quality assessment of RNA

The Bioanalyser 2100 (Agilent Biotechnologies Inc., Santa Clara, CA, USA) was used to assess the quality of the RNA using a RNA 6000 Nano Chip Kit (Agilent Biotechnologies). A RNA Integrity Number (RIN) is returned. In this study samples with a RIN score lower than 6 were excluded.

Normalisation, dilutions and sample sorting

The Hamilton robot was used to normalize the DNA to either a concentration of 50ng/µl or 20ng/µl. All DNA was stored in the UK DNA Companion Animals archive at either -40ºC or -80ºC. The XL20 robot was used to cherry pick samples for the study using 2D barcodes on each sample tube. Finally the Cybio robot was used in some PCR protocols to dilute DNA or to add SAP solution to PCR products.

RNA Microarray

The canine microarray was designed and preformed by WALTHAM® Centre for Pet Nutrition; details of the assay are summarised in Chapter 3.

36 Q PCR assay design

The qPCR assays were all performed in triplicate using a TaqMan™ ABI PRISM 7900 SDS (Applied Biosystems, Foster City, CA, USA) in a 384-well plate format. A 10-μl reaction volume was used per well. This consisted of 5 μl Taqman 2X PCR master mix (Universal PCR Mastermix; Applied Biosystems), 0.1 μl each of 20 μM forward and reverse primers (MWG Biotech, Eurofins, Germany) 0.1 μl of 10 μM probe (Exiqon; Roche

Diagnostics Ltd), 0.1 μl distilled water and 4.6 μl of sample cDNA or water for the negative controls.

The amplification was performed according to the standard ABI 7900 protocol: 10 minutes at 50°C followed by 40 cycles of 95°C for 1 minute and 60°C for 15 seconds, as recommended by the manufacturer (Applied Biosystems). The real-time data were analysed by Sequence Detection Systems software, version 2.2.1 (Applied Biosystems). The detection threshold was set manually at 0.05 for all assays. Standard curves were generated for each assay, to confirm the efficiency of the assay is between 93%-107% and the R2 value was > 0.98.

Touchdown PCR

Touchdown PCR was used as opposed to traditional PCR because it reduces non-specific priming and therefore the background „noise‟ caused by non-specific DNA fragments is reduced. It does this by gradually lowering the annealing temperature as the PCR cycles progress. Initially the annealing temperature is set 7°C above the temperature of melting (Tm) of the primers used, at each cycle the temperature is reduced by 0.5°C. The initial higher

temperature increases specificity, and the subsequent lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.

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The touchdown PCRs were performed throughout to optimize primers and were on either a DNA engine Dyad™ peltier thermal cycler or a DNA engine Tetrad™ PTC-225 peltier thermal cycler (MJ research Inc., Waltham MA, USA) using these cycling conditions: 95ºC for 5minutes, followed by 95ºC for 30seconds, Ta+7ºC for 30 seconds (reduce by 0.5ºC per cycle) and 72ºC for 1minute for 13 cycles, and 95ºC for 20 seconds, Ta for 1 minute, and 72ºC for 1 minute for 19 cycles, and finally 72ºC for 5minutes.

Gradient PCR

Gradient PCR was used when touchdown PCR failed to optimise primers, this allowed a temperature range to be run across the primers establishing the correct temperature for amplification. Once this temperature was established touchdown PCRs were performed.

Electrophoresis-gel quality check

All PCR products were run on a 2% agarose gel with a 1kb ladder (Promega, Southampton, UK) to assess the concentration and size of the PCR product.

Transgenomic WAVE

Details of sample selection, primer design, cycling conditions and equilibration are in Chapter 6. Conditions were generated for each fragment using WAVEMAKER V4.1.44 (Transgenomic, Huston, Texas, USA) software.

Microsatellite genotyping

The initial PCR assays were all performed on the Dyad™ peltier thermal cycler or a DNA engine Tetrad™ PTC-225 peltier thermal cycler (MJ research Inc., Waltham MA, USA) in a 384-well plate format. A 10μl reaction volume was used per well: 0.04μl Hot start Taq (5U/µl Qiagen, Crawley, UK), 0.05μl each of 50pmol/µl forward (labelled) and reverse

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primers (Metabion, Germany), 0.3µl MgCl2 (25mM, Qiagen, Crawley, UK), 0.5μl DNTPs

(2mM), 1µl x10 buffer (Qiagen, Crawley, UK) 3.06µl distilled water and 5µl of DNA at 5ng/µl. The cycling conditions were as follows: 95ºC for 5 minutes, then 94ºC for 20 seconds, 55ºC for 30 seconds and 72ºC for 30 seconds for 10 cycles, 89ºC for 20 seconds, 55ºC for 30 seconds and 72ºC for 30 seconds for 20 cycles and finally 72ºC for 10 minutes. ABI prism® Rox400 (Applied biosystems, Foster City, CA, USA) was used for accurate sizing. The ABI prism® 3100 genetic analyzer data collection software V1.1 (Applied biosystems, Foster City, CA, USA) was used to set up the run. The ABI prism® Genescan software V3.7 (Applied biosystems, Foster City, CA, USA) was then used to analyse the data. ABI prism® Genotyper software V3.7 (Applied biosystems, Foster City, CA, USA) was used to group the results into genotypes/microsatellites. Full details are in Chapter 6.

Illumina canine SNP20 GWAS array

The Illumina canine SNP20 GWAS array was performed by the Barts and the London Genome Centre according to the manufacturers‟ instructions; assay details are in Chapter 7.

Sequenom genotyping

Sequences of genes to be genotyped were retrieved from Ensembl using the Biomart data mining tool156 and exported in FASTA format. A PERL script was used to convert the FASTA file into a suitable format for the Sequenom design tools. Using ProxSNP and Prextend157 quality control procedures were run on the sequences applied, rejecting

unsuitable sequences for the Sequenom platform. The SNPs were then plexed together by the assay design software (MassARRAY) in a way that means the greatest chance of genotyping success and the largest number of SNP possible was included on each plex.

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All primers and probes were supplied by Metabion (Germany); the probes were diluted using distilled water to 400µM and the primers to 100µM using the Hamilton Robot. The primers were then pooled together in plexes, each primer within the pool at a concentration of 500nM done by the Hamilton robot. Probes were also pooled into plexes although the plex was split into low mass and high mass probes; low mass at a concentration of 7µM and high mass at 14µM, again performed on the Hamilton robot. The primers and probes were then ready for PCR, details of which are given in Chapters 8 & 9.

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Chapter 3

Candidate Genes & Microarray analysis

of Gene Expression

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