Nanodrop

Total RNA was extracted from the prepared plant material using an RNAqueous^® Total RNA Isolation kit (Thermo Fisher Scientific Baltics UAB, AM1912, Vilnius, Lithuania). The extracted RNAs were first treated with DNaseI using a DNAfree Kit (Thermo Fisher Scientific Baltics UAB, AM1906, Vilnius, Lithuania). RNA quantity was checked using a NanoDrop and quality was tested in 1.5% agarose gel (Sigma-Aldrich, J2500, St. louis, MO, USA). cDNA was synthesized by reverse transcribing 1 μg RNA using a SuperScript II Reverse transcriptase kit (Thermo Fisher Scientific, 18064-014) with anchored-oligo (dT)₁₈ primer (Thermo Fisher Scientific, SO132) according to the manufacturer’s instructions.

RNA was isolated as mentioned above, quantified using a Nanodrop and DNAse treated (DNAse kit, Sigma AMPD1). The RNA was then reverse transcribed into cDNA using the High‐Capacity cDNA Reverse Transcription kit (Applied Biosystems, 4368814) following the manufacturer's recommendations. RNA samples were collected from three independently isolated “biological replicates,” and three “technical replicates” of each strain/condition were tested for a given biological replicate. Quantitative PCRs were conducted using the PowerUp SYBR Green Master Mix kit (Applied Biosystems A25741) on the CFX Connect Machine (BioRad). Gene expression data were analyzed using the ΔΔC_t method and normalized to the housekeeping gene, rpl‐32. Melt curves for all reactions were run confirming the integrity of the reaction. Primer sequences are included in Table S11.

Total RNA was extracted from fibroblasts from patients with CD on a GFD and controls using TRIZOL reagent (Ambion®-Life Technologies). The mRNA concentration was measured using a Nanodrop® spectrophotometer, and subsequently, the RNA quality was analyzed using agarose gel electrophoresis in Tris/Borate/EDTA buffer (TBE, Sigma, Milan, Italy), as described previously^{18 (link)}. The RNA (1 µg) was reverse transcribed into cDNAs using the High Capacity cDNA Reverse Transcription kit, according to the manufacturer’s protocol. Experiments were performed with approximately 40 ng of cDNA templates, according to the manufacturer’s protocol (TaqMan® Gene Expression Assay), using a 7900HT Fast Real-Time PCR system. The gene expression assay used for EEA1 gene is Hs00929215_m1 (Life Technologies), and the probe is located on Chr 12 exons 28–29 (caaga ttggatctac agaaaaaatc tgaagccctt gaaagtatca agcaaaagct taccaagcaa gaggaagaaa aacaaatcct gaaacaagat tttgaaactt taagtcaaga aacaaagatt). The expression of each gene was normalized to the expression of an endogenous housekeeping gene (B2M). Relative quantification was performed using the ΔΔCt method. SDS software (ABI, version 1.4 or 2.4) was used to analyze the raw data, and subsequently, an additional statistical analysis was performed using GraphPad Prism 5.01® software.

Total RNA was isolated from homogenized mouse lung using Tri Reagent (Sigma) and quantified by NanoDrop (Nd-1000). Reverse transcription was performed withSuperScript III Kit according to manufacturers’ instructions (Invitrogen). cDNA was subjected to quantitative PCR using primers for Muc5ac (sense 5′-CAGCCGAGAGGAGGGTTTGATCT-3′ and anti-sense 5′-AGTCTCTCTCCGCTCCTCTCA-3′; Sigma). Relative transcript expression of a gene is given as 2−ΔCt(ΔCt = Cttarget−Ctreference), and relative changes compared with control are 2−ΔΔCtvalues (ΔΔCt = ΔCttreated−ΔCtcontrol) {John, 2014 #340}.

RNA from heart tissue was extracted using TRIzol (Sigma-Aldrich) and quantified using the Nanodrop as reported previously (4 (link)).