Total RNA was isolated from the HUVECs (2 × 106 cells/well) using the TRIZOL reagent (15596018, Invitrogen, CA, United States) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg of total RNA using the iScript gDNA Clear cDNA Synthesis Kit (170–8890, Bio-Rad Laboratories, Redmond, United States). Quantitative RT-PCR was performed using a Fast EvaGreen Supermix (172-5260, Bio-Rad Laboratories) on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories) following the manufacturer’s protocol. The following optimized PCR conditions were used: 95°C for 30 s, 95°C for 5 s, and 40 cycles at 60°C for 5 s. The mRNA levels of the target genes were normalized to the endogenous GAPDH levels, and the expression levels of the target genes were analyzed using the 2−ΔΔct method. Table 1 presents all the primers used for qRT-PCR.
Fast evagreen supermix
Manufactured by Bio-Rad
Sourced in United States
Fast EvaGreen Supermix is a ready-to-use solution for real-time PCR applications. It contains all the necessary components, including the EvaGreen dye, for efficient and sensitive DNA amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Light cycler 480 instrument rt pcr detection system, by Roche (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Iscript gdna clear cdna synthesis kit, by Bio-Rad (1 mentions)
Rnaeasy, by Qiagen (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
7 protocols using fast evagreen supermix
1
Quantitative Gene Expression Analysis in HUVECs
2
Osteoblast Progenitor Gene Expression
3
Gene Expression Analysis by qRT-PCR
4
GTN-Induced Changes in DDAH-1 mRNA Expression
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Rats (N = 6 per experimental group) were injected with GTN (10 mg/kg i.p.) or vehicle and then killed with a lethal dose of anaesthetic 4 h after treatment. Their brains were immediately chopped into parts and frozen at −80 °C until further processing.
DDAH-1 mRNA expression was analyzed by a real-time polymerase chain reaction (RT-PCR) and total RNA was isolated from the cerebral samples with Trizol reagent in accordance with the method of Chomczynski and Mackey [26 (link)]. RNA was quantified by measuring the absorbance at 260/280 nm. cDNA was generated using the iScript cDNA Synthesis kit (Bio-Rad) following the supplier's instructions. Gene expression was analyzed using the Fast Eva Green supermix (Bio-Rad). As regards housekeeping, gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used. The expression of the housekeeping gene remained constant in all the experimental groups considered. The amplification was performed through two-step cycling (95–60 °C) for 45 cycles in a light Cycler 480 Instrument RT-PCR Detection System (Roche) following the supplier's instructions. All samples were assayed in triplicate. Gene expression was calculated using the ΔCt method.
DDAH-1 mRNA expression was analyzed by a real-time polymerase chain reaction (RT-PCR) and total RNA was isolated from the cerebral samples with Trizol reagent in accordance with the method of Chomczynski and Mackey [26 (link)]. RNA was quantified by measuring the absorbance at 260/280 nm. cDNA was generated using the iScript cDNA Synthesis kit (Bio-Rad) following the supplier's instructions. Gene expression was analyzed using the Fast Eva Green supermix (Bio-Rad). As regards housekeeping, gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used. The expression of the housekeeping gene remained constant in all the experimental groups considered. The amplification was performed through two-step cycling (95–60 °C) for 45 cycles in a light Cycler 480 Instrument RT-PCR Detection System (Roche) following the supplier's instructions. All samples were assayed in triplicate. Gene expression was calculated using the ΔCt method.
5
Gene Expression Analysis of Cannabinoid Receptors in PBMCs
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Following the manufacturer’s instructions, total RNA from PBMCs was isolated using the standard methods (Zymo Research, Roma, Italy), and the quality of the RNA was evaluated using a NanoDrop spectrophotometer (NanoDrop Technologies, Thermofisher, Waltham, MA, USA). cDNA was then produced using the iScript cDNA Synthesis kit (Bio-Rad, Milan, Italy). Using the Fast Eva Green supermix, we assayed the gene expression of CB receptors, FAAH, MAGL, NAPE-PLD and DAGLα (Bio-Rad).
Table 4 lists the primer sequences that were acquired using the AutoPrime program http://www.autoprime.de/AutoPrimeWeb (accessed on 18 December 2023). As a housekeeping gene, ubiquitin C (UBC), whose expression was constant across all experimental groups, was employed. Following the supplier’s instructions, the amplification was carried out using a light Cycler 480 Instrument rt-PCR Detection System (Roche). All samples were assayed in triplicate and gene expression levels were calculated according to the 2−ΔΔCt = 2−(ΔCt gene − ΔCt housekeeping gene) formula by using Ct (cycle threshold) values.
6
Quantifying Cytokine Expression in Monocytes
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Total RNA from isolated monocytes was obtained using a standard procedure (Zymo Research) and RNA quality was assessed using a spectrophotometer (Nanodrop™ Thermo Fisher Scientific); cDNA was generated using an iScript cDNA Synthesis Kit (Bio-Rad) following the supplier’s instructions. The gene expression of pro-inflammatory and anti-inflammatory cytokines was analyzed using the Fast Eva Green Supermix (Bio-Rad). The primer sequences obtained from the AutoPrime software (http://www.autoprime.de/AutoPrimeWeb ; accessed on 4 December 2017) are reported in Table 2 . Ubiquitin C (UBC), whose expression remained constant in all experimental groups, was used as housekeeping gene. The amplification was performed with a light Cycler 480 Instrument rt-PCR Detection System (Roche) following the supplier’s instructions. All samples were assayed in triplicate and gene expression levels were calculated according to the 2−∆∆Ct = 2−(Ct gene − Ct housekeeping gene) formula by using Ct values.
7
Ferret Gene Expression Analysis
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Primers were designed by amplification of ferret FSHβ sequences (GenBank No. NW_004569193), NCOA1 sequences (GenBank No. NW_013052970), and β-Actin (GenBank No. XM_004775013) in Table 2. Total RNA was isolated from tissue samples using the Extraction RNA Kit (Takara, Japan). Dried RNA samples were dissolved in diethylpyrocarbonate-treated water. The RNA integrity was verified by agarose gel electrophoresis and visualization of 28S and 18S ribosomal RNA. Reverse transcription was performed with 2 μg of total RNA using the Prime Script RT Master Mix Perfect Real Time reaction kit (Takara Biotechnology Co., Ltd., Dalian, People's Republic of China). Real-time PCR was performed in triplicate in a 10 μL volume containing 1 μL of cDNA, 0.3 μL of reverse and forward primers for each gene, 3.4 μL of double-distilled water, and 5 μL of Fast Eva Green Supermix (Bio-Rad, Hercules, CA, USA). Amplification conditions were 94 °C for 5 min, followed by 40 PCR cycles of 20 s at 94 °C, and 30 s at 63 °C. Expressions of NCOA1 and FSHβ were quantified in duplicate and normalized to the housekeeping gene β-Actin. Melt curve analysis was performed and the relative abundance of NCOA1 and FSHβ mRNA was calculated by the 2 -ΔΔCT method.
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