qRT-PCR was performed on each cDNA replicate in duplicate (i.e. 4 technical replicates in all), using TaqMan Universal PCR Master Mix, No AmpErase UNG (Life Technologies, cat. no. 4324018) and an appropriate TaqMan mRNA/pre-mRNA assay (Life Technologies).
Assay imprecisions, evaluated by measurement of 6 replicates in duplicate from a serum pool using three different cartridges, were less than 10%% for all 4 cytokine protein biomarkers, comparable to the imprecisions of traditional ELISAs.
To determine the expression stability of the selected genes in B. oleae head, the expression of the reference genes was measured in 24 heads (6 individuals from each of the LAB, SPIN, w-GR and w-CAL populations, i.e., 24 biological replicates) in duplicate reactions (two technical replicates).
For the quantitative analysis, three biological replicates in duplicate (n = 6) were stained with colloidal Coomassie Brilliant Blue G-250 (cCBB; [ 56]) and analyzed with ImageMaster 2-D Platinum Software GE Healthcaree) in order to quantify the spots (%Vol: percentage of the total spot volume) assigned to GS by WB.
Additionally, 6 of the 24 samples were replicated in the mRRBS study as analytic duplicates.
Real-time PCR reactions were carried out in duplicate and replicated in a minimum of three independent experiments.
To evaluate reproducibility, tumour samples that were found to contain a significant mutation were replicated in at least duplicate.
PCR reactions (three biological replicates in duplicates) were performed with 0.5 ng cDNA on a Stratagene MX3000P thermocycler (qPCR MxPro v4.01) using the Absolute qPCR SYBR green ROX mix (ABgene).
Semi-quantitative PCR was performed on at least three biological replicates measured in duplicates for each gene, and non-template controls were included.
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