Nanodrop 3300 spectrophotometer

Following experiments, endothelial cells were harvested for extraction of total RNA according to the method of Chomczynski and Sacchi [26] (link) using TRIzol reagent (Life Technologies, Dublin, IRL). cDNA was generated from 1 µg of total RNA using a high capacity cDNA reverse transcription kit (Life Technologies). Quantitation of the final cDNA was determined using a NanoDrop 3300 Spectrophotometer (Thermo Scientific, DE, USA). Amplification of target cDNA sequences using gene-specific primers was subsequently performed and analyzed as previously described [27] (link). Ribosomal subunit S18 was routinely used for normalization purposes. Primer pairs (shown below) were screened for correct product size (1% agarose gel electrophoresis) and underwent melt-curve analysis for primer-dimers. TM (107 bp): Forward 5′-ACCTTCCTCAATGCCAGTCAG-3′; Reverse 5′-GCCGTCGCCGTTCAGTAG-3′; S18 (250 bp): Forward 5′-CAGCCACCCGAGATTGAGCA-3′; Reverse 5′- TAGTAGCGACGGGCGGTGTG-3′.

TRIzol reagent (Invitrogen) was utilized to isolate total RNA from NCP tissues and cells. The RNeasy FFPE kit (QIAGEN GmbH) was utilized to extract total RNA from FFPE NPC tissues. The RNA quality and amount were evaluated by a NanoDrop 3300 spectrophotometer (Thermo Scientific). Reverse transcriptase (Promega) was utilized to perform reverse transcription. SYBR Green qPCR SuperMix-UDG (Thermo Fisher) was utilized to conduct Quantitative real-time PCR. β-actin was used as the normalization control. Specific primers are shown in Supplenentary Table S1.

For total RNA isolation pieces of frozen samples were homogenized in TissueLyzer (QIAGEN) for 8 min at 50 Hz in Extract RNA reagent (Evrogen). Next purification steps were performed following instructions for Extract RNA reagent. Quality and purity of RNA were validated using NanoDrop 3300 SpectroPhotometer (Thermo Fisher Scientific) and electrophoresis in 1% agarose gel. RNA samples were treated with DNAseI (Thermo Fisher Scientific). cDNA template was synthesized using Random (dN)10-primer (Evrogen) and MMLV RT kit (Evrogen). Quantitative Real-Time PCR (qRT-PCR) was performed on cDNA template using commercial TaqMan Gene Expression Assays for Nppa, Fhl1, Fhl2, Actn2, Synpo2, Myoz2, Nebl, Cmya5, Ldb3, Csrp3, Ilk, and Actb genes (Applied Biosystems). Expression of Hprt1 was evaluated by qRT-PCR using oligonucleotide gene-specific primer pair. The list of TaqMan assays and primers is provided in Table 1. mRNA relative expression was quantified applying _ΔΔC_t-method using Actb and Hprt1 as housekeeping control (Livak and Schmittgen, 2001 (link)).

DNA and RNA were isolated from each sample, and their purities were measured; their quality was based on RIN, DIN, and DV₂₀₀. DNA was isolated using a QIAamp DNA formalin-fixed, paraffin-embedded (FFPE) Tissue Kit (Qiagen, Hilden, Germany). RNA was isolated using an miRNeasy FFPE Tissue Kit (Qiagen). The purity of the nucleic acid samples was assessed based on their spectrophotometric absorbance at 260–280 nm (A260/A280) using a NanoDrop 3300 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RIN, DIN, and DV₂₀₀ indices and the concentrations of the extracted DNA and RNA samples were quantified using TapeStation 4150 (2015, Agilent Technologies, Santa Clara, CA, USA) based on the genomic DNA (gDNA) and RNA ScreenTape assays. Owing to instrument performance, RIN, DV₂₀₀, and DIN can provide quality measurements in samples with RNA concentrations >2.0 ng/μL and gDNA concentrations >3.0 ng/μL. Statistical analysis was performed on cases with evaluable RIN, DV₂₀₀, and DIN. Subsequently, to asses DNA quality and detect variants, NGS was conducted using an MiSeq sequencing platform. The AmpliSeq for Illumina Cancer HotSpot Panel v2 (50 target genes, 207 amplicons; Illumina Inc., San Diego, CA, USA) protocol was applied (Supplemental Information S5).

Total RNA was isolated from different broccoli samples using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The quantity of RNA was measured using a NanoDrop 3300 spectrophotometer (Thermo Scientific). One microgram of the total RNA was reverse-transcribed with Superscript™ III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The resulting cDNA samples were diluted to 1/10 their concentrations (v/v) for qRT-PCR. The list of the primers used can be found in Supplementary Table S1. The primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Quantitative real-time PCR was carried out with the Power SYBR^® Green RT-PCR Master Mix (Qiagen) using a QuantStudio 3 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The relative expression ratio was determined with the Equation 2^−∆∆Ct using the BoACT2 normalized ∆Ct values generated by the QuantStudio 3.

Dynamic light scattering (DLS) measurements were performed on a Zetasizer-nano (Malvern Instruments Ltd., Worcestershire, UK) to determine the particle size distribution. Transmission electron microscopy (TEM) was performed using a JEM-1400 microscope (JEOL Ltd., Tokyo, Japan) operating at 120 kV. The samples for TEM were dispersed in purified water and transferred onto 400 mesh size copper grids coated with a thin film of collodion and carbon, followed by staining with uranyl acetate. Fluorescence intensity was measured using a NanoDrop 3300 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).