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As a result, the EF1α gene showed the most stable expression pattern in all multiplex plates (data not shown).
Multiplex plates were analyzed using a Luminex 100 instrument with StarSection software (Applied Cytometry Systems, Dinnington, UK).
Enzyme-linked immunosorbent assays (ELISA) using multiplex plates (Millipore, Billerica, MA, USA) were performed on a subset of the population (18 lean and 31 obese).
Out of the 99 genes represented on multiplex plates and Affymetrix microarrays (among the 18,041 probe sets retained after the filtering step), 85 genes (86%) displayed concordant results according to the two approaches – whereas only 14 genes showed discordant results.
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Multiplex plate-based sandwich ELISAs, such as the MSD Mesoscale® (Gaithersburgh, MD, USA) and SearchLight® (Fishers Scientific, Pittsburgh, PA, USA) chemiluminescent arrays are capable of quantification of up to 16 different proteins in multi-well plates.
We used the ultrasensitive Th1/Th2 cytokine multiplex plate to measure IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-6 and TNF-α (Meso Scale Discovery, Rockville, USA) according to the manufacturer's recommendations.
EDTA blood samples were collected at baseline (T0) and after 4 weeks (T1) of treatment, together with radiological imaging in all 41 patients to analyse plasma circulating angiogenic factor levels by multiplex ELISA plates.
Over the years, improvements in these platforms as well as newer technologies such as multiplex RT-PCR plates, RNA-sequencing and Nanostring have enabled an in-depth characterization of microRNome changes in many cancer types [ 52 55].
Serum cytokine levels were analysed using BioRad multiplex cytokine magnetic plate assay (BioRad).
RNA libraries are prepared according to the TruSeq RNA Sample Preparation v2 protocol, and the DNA libraries are applied to the cluster generation and sequencing using c-BOT Multiplex re-hybridization plate and Truseq Sbs kit V3.
Millipore multiscreen 96-well filter plates for multiplex cytokine kits were used.
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