Microarray hybridisation and data analysis
Four biological replicates per genotype were used for RNA extraction
using the method described above. RNA (200 ng) was used for cDNA
synthesis and Cy3-labelling using the Low Input Quick Amp Labelling Kit
for One-Colour Microarray-Based Gene Expression Agilent analysis
(Agilent, Santa Clara, CA, USA). Linearly amplified and labelled cDNA
(1.65 µg) was hybridised for 17 h at 65ºC on 4 X 180 k format 60-mer
oligonucleotide probes designed against the S. lycopersicum cv.
Heinz 1706 build SL2.40 (annotation 2.3) genome (Agilent design ID =
069672; see Gene Expression Omnibus (GEO) record GPL21602). Each array
contained ~5 probes for 34,619 transcripts. Arrays were
imaged using an MS200 microarray scanner using only the 480 nm laser
using the autogain feature of the NimbleScan software (Roche NimbleGen,
Madison, WI, USA). Image (tiff) files were imported into the Agilent
Feature Extraction software for quality control assessment, grid
alignment and expression value extraction at the probe and transcript
level with the RMA algorithm (Irizarry et al. 2003) used to carry
out background subtraction, quantile normalisation and summarisation via
median polish, and output log2 normalised gene expression levels (GEO
record GSE79307)(Ferrández-Ayela et al. 2016). Linear Models for
Microarray Data (package LIMMA in R) was then used to fit linear models
to pairs of samples, identifying genes that contrasted the most between
the experimental pairs (Smyth 2004). Transcripts were deemed to be
differentially expressed if they showed a Benjamini-Hochberg adjustedP ≤ 0.05 when comparing rootstocks genotypes.