Rt primer mix

The expression of 84 genes related to endothelial cell biology (catalogue number PAHS-015ZD-24, Qiagen, Hilden, Germany) by HUVEC was determined using QuantiTect Primer Assay (Qiagen, Hilden, Germany) in a real-time polymerase chain reaction (RT-PCR) as previously published [2 (link)]. Briefly, total RNA from HUVEC pretreated with DBMSCs and 100 μM H₂O₂ for 48 h was isolated, and cDNA was then synthesized using FastLane Cell cDNA kit and RT Primer Mix (Qiagen) as previously published [2 (link)]. After quantifying mRNA using QuantiTect SYBR Green PCR Kit (Qiagen), the real-time PCR reaction was performed in triplicate on the CFX96 real-time PCR detection system (BIO-RAD) as previously published [2 (link)]. To analyze the data, the CFX manager software (Bio-Rad, CA, USA) was used. The results were exported to Microsoft Excel for further analysis. The results were expressed as fold change by calculating the ΔΔ⁻² values. The relative expression of an internal house-keeping gene as a loading control was used as provided in the kit. Experiments were performed in triplicate using HUVEC prepared from independent umbilical cord tissue and repeated three times.

Total RNA was isolated from algal cultures grown to late-exponential phase with the Direct-Zol^TM RNA MiniPrep kit (Zymo Research, Freiburg, Germany) followed by an additional DNase treatment using TURBO DNase (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA yield and quality were assessed by determining the 260 nm/280 nm absorbance ratio using a Nanodrop instrument (Thermo Fisher Scientific).
Unless otherwise stated, first-strand cDNA was synthesized from total RNA (1.7–4 µg) using the RT primer mix (Qiagen) and Superscript III reverse transcriptase (Invitrogen) following the manufacturer´s protocol. Subsequently, the RNA was removed by RNase H (Invitrogen) treatment. The absence of contaminating genomic DNA was confirmed by the inclusion of control samples that had not undergone reverse transcription. In order to determine the transcript ratio of the srta-1 mutant allele and the SRTA wild-type allele in the complemented lines of strain UVM11, the SRTA-specific primer SRT3402r2 (5′- GCTCGCCGGTCCTTGG-3′) was used to prime the reverse transcription reactions. The sequence containing the srta-1 point mutation was amplified with primers SRTE5f (5′-AGCTGGTGGACAACATCCTG-3′) and SRT8r (5′-GCGTCTTCTGCAGGTTCAC-3′).

Delta-9-tetrahydrocannabinol (∆⁹-THC) was obtained from the National Institute on Drug Abuse Drug Supply (Bethesda, MD). For all experiments, ∆⁹-THC was dissolved in 0.9% saline, 5% Cremaphor EL, and 5% ethanol (18:1:1 v/v/v) and administered intraperitoneally (IP) in an injection volume of 10 ml/kg, 60 minutes prior to testing. Doses of Δ⁹-THC were selected based on previous data obtained in our lab that resulted in a 70% maximum possible effect (%MPE) in the tail-flick assay in male mice (Henderson-Redmond et al. 2020 (link), 2021 (link)). An additional group of mice was treated with vehicle (VEH) alone to serve as a control group. VEH was prepared using 0.9% saline, 5% Cremaphor EL, and 5% ethanol (18:1:1 v/v/v) and given by IP injection of 10 ml/kg 60 minutes prior to testing. RNAse Zap, Buffer RW1, Buffer RPE, diethyl pyrocarbonate water (DEPC H₂O), Wipeout Buffer, Quantiscript^® Reverse Transcriptase (RT), Quantiscript RT Buffer, RT Primer Mix, and Rnase-free water were obtained from Qiagen, Trizol from Thermo Fisher Scientific, and chloroform from Lab Alley. The Primetime Gene Expression Master Mix, rox reference dye, and Taqman primers (CB₁, CB₂, and β-actin) are from IdT Technologies.

RNA from the 27 transgenic Arabidopsis samples and the 15 pear samples (fully expanded mature leaves) used for anthocyanin extractions were extracted by using Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, United States). First-strand cDNA synthesis was carried out by using RT Primer Mix according to manufacturer’s instructions (QuantiTect Reverse Transcription kit, Qiagen, Hilden, Germany). Real-time qPCR DNA amplification and analysis was carried out using the LightCycler 480 Real-Time PCR System (Roche Diagnostics, Mannheim, Germany), with LightCycler 480 software version 1.5. The LightCycler 480 SYBR Green I Master Mix (Roche) was used, and 10 μl of total reaction volume applied in all the reactions following the manufacturer’s method. qPCR conditions were 5 min at 95°C, followed by 40 cycles of 5 s at 95°C, 5 s at 60°C, and 10 s at 72°C, followed by 65–95°C melting curve detection. The qPCR efficiency of each gene was obtained by analysing the standard curve of a cDNA serial dilution of that gene. Sequence of the primers used are provided in Supplementary Table S1.

In each reverse transcriptase reaction, 150–300 ng of purified RNA was used to generate cDNA. Following genomic DNA elimination with gDNA wipe-out buffer (Qiagen, Germantown, MD, USA) at 42°C for 2 min, cDNA was prepared by setting up an RT-PCR reaction with RNA template, Quantiscript reverse transcriptase, Quantiscript RT buffer, and RT primer mix (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions.
Quantitative PCR (qPCR) reactions were set up with 1× SSo Advanced™ Universal SYBR^® Green Supermix (Bio-Rad, Mississauga, ON, Canada), 250–500 nM of forward and reverse primers (Qiagen, Germantown, MD, USA or Bio-Rad, Mississauga, ON, Canada), and nuclease-free water in Bio-Rad CFX96 touch real-time PCR detection system. qPCR data were analyzed using CFX Maestro™ software. Gene expression values were determined using the formula ΔCq = Cq of the gene of interest − Mean of Cq of housekeeping genes, and normalized expression was then calculated as 2^−(ΔCq). A predesigned 384-well PCR panel (Bio-Rad, acute inflammation response H384) was used to screen uninfected vs. C. trachomatis-infected mast cell gene responses according to the manufacturer’s instructions.