Quantiscript rt buffer

Total RNA was isolated with Trizol from tumor as well as normal tissue samples collected from each patient and then purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Synthesis of cDNA was performed with 1 μg of total RNA from each sample using Quantiscript Reverse Transcriptase, Quantiscript RT-buffer and RT Primenr-mix of QuantiTect Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using in 96-well optical reaction plates (Applied Biosystems, Darmstadt, Germany) using a StepOnePlus amplification and detection system (Applied Biosystems). The real-time RT-qPCR reactions were prepared using SYBR® Select Master Mix (Life Technologies), and the following conditions were used: 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The primers of target genes used for this analysis were Bim and PUMA, and GAPDH was used as the reference gene. The primer sequences are listed in Table S3. The gene copy numbers of Bim and PUMA were calculated by using a standard curve that was constructed using the OE33 cell line. The 2^−ΔΔCT method was used as a relative quantification strategy for qPCR data analysis. In total, 20 samples from Ile/Ile, 12 from Ile/Val and 6 from Val/Val genotype were used in this study.

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.

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.