Nanodrop 2000 2000c instrument

On the patient’s arrival at the hospital for admission, blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes. Subsequently, the plasma was separated, and the presence of hemolysis in the plasma was evaluated through a first visual inspection and a second review based on spectrophotometry with the NanoDrop™ 2000/2000c instrument (Id: ND-2000, Thermo Scientific™, Foster City, CA, USA) with readings at wavelengths from 350 to 650 nm. Samples are considered hemolyzed if the reading at 414 nm exceeds a value of 0.2 [60 (link),61 (link)]. Samples that did not meet this requirement were discarded for this study. Afterward, RNA extraction was performed with the miRNeasy serum/plasma Advanced Kit (Qiagen, Hilden, Germany). For normalization, synthetic Caenorhabditis elegans 39-3p (cel-miR-39-3p) was added as an external reference miRNA (1.6 × 10⁸ copies/μL). The next step was quantifying the RNA obtained via spectrophotometry at 260 nm wavelength using the NanoDrop™ 2000/2000c instrument (Id: ND-2000, Thermo Scientific™, Foster City, CA, USA). The contamination with organic compounds and proteins was established using the ratio of the readings 260/230 and 260/280, respectively; the samples were considered free of contaminants when the relationship was between 1.7–2.0. Then, they were stored at −20 °C until use.

Total RNA was isolated from human normal colon and UC patient tissues using TRI-Solution (BioScience Technology, Rockaway, NJ, USA) following the manufacturer’s protocol. RNA quantity was measured using a NanoDrop 2000/2000c instrument (Thermo Scientific, Waltham, MA, USA) and 1 µg of total RNA was reverse-transcribed into cDNA using the iScript^TM cDNA Synthesis kit (BioRad, Hercules, CA, USA). For expression studies, primers were designed using the Primer3 web tool (http://frodo.wi.mit.edu/primer3), and listed in Table S2. Quantitative RT-PCR was performed on a CFX96^TM Real-Time PCR Detection System (Bio-Rad) using a Syber Green master mix (Thermo Scientific, Waltham, MA, USA). The expression levels of target genes were normalized by β-actin levels, and all relative quantifications of expressions were calculated using the ΔΔC_t method.

Total RNA was isolated from the control and irradiated samples of HAECs and HCAECs using TRI-Solution (BioScience Technology) following the manufacturer’s protocol. RNA quantity was measured using a NanoDrop 2000/2000c instrument (Thermo Fisher Scientific), and 1 μg of total RNA was reverse-transcribed into cDNA using the iScript™ cDNA Synthesis Kit (Bio-Rad). For the expression studies, primers were designed using the Primer3 web tool (http://frodo.wi.mit.edu/primer3) and are listed in Table S1 (Additional file 1). Quantitative real-time reverse transcription PCR (qRT-PCR) was performed on a CFX96™ Real-Time PCR Detection System (Bio-Rad) using SYBR Green Master Mix (Thermo Fisher Scientific). The expression levels of target genes were normalized against actin levels, and all relative quantifications of expression levels were calculated using the ∆∆C_t method.