Total RNA was isolated from cells by using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. First-strand cDNA synthesis was performed using reverse transcription kit (Toyobo, Japan) and quantitative real-time PCR (qRT-PCR) was performed with SYBR Green Supermix Taq Kit (Takara, Dalian, China). All reactions were carried out in triplicate. Relative gene expression was calculated by using ΔΔCt method. All the qRT-PCR primers used are listed in Table 2 .
Sybr green supermix taq kit
Manufactured by Takara Bio
Sourced in China
The SYBR Green Supermix Taq Kit is a reagent solution designed for real-time PCR (polymerase chain reaction) applications. The kit contains a DNA polymerase, buffer, and SYBR Green I dye, which specifically binds to double-stranded DNA and emits fluorescent signals that can be detected and quantified during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Iq5 real time pcr detection system, by Bio-Rad (2 mentions)
Reverse transcription kit, by Toyobo (1 mentions)
Myiq two color real time pcr detection system, by Bio-Rad (1 mentions)
Cdna rt kit, by Toyobo (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Icycler, by Bio-Rad (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rabbit polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit antibody, by Beyotime (1 mentions)
Real time pcr detection system, by Bio-Rad (1 mentions)
First strand cdna synthesis kit, by Toyobo (1 mentions)
5 protocols using sybr green supermix taq kit
1
Quantitative RT-PCR for Gene Expression Analysis
2
Quantifying Mesenteric Artery Gene Expression
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Total RNA from 10 animals each group was isolated from mesenteric arteries using Tri-Reagent (Invitrogen, CA, USA) and reverse-transcribed to cDNA (cDNA RT kit; Toyobo, Japan). Real-time PCR was performed with SYBR Green detection (SYBR Green Supermix Taq Kit, Takara, Japan) on iCycler, MyiQ two Color Real-Time PCR Detection System (Bio-Rad) according to the manufacturer's instructions. The gene specific primers (Sangon, Shanghai, China) were shown in Table 1 . All primers were verified to yield a single PCR product with the correct molecular weight. The PCR conditions were 5 minutes at 95°C, 40 cycles of 15 seconds at 95°C followed by 15 seconds at 60°C and 30 seconds at 72°C. ΔΔCt method was used to comparatively quantify the amount of mRNA.
3
Quantifying Placental Vessel RNA Levels
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Total RNA was isolated from placental vessels using Trizol reagent, and was reversed transcribed using the first‐strand cDNA Synthesis Kit (Invitrogen Corp., Carlsbad, CA, USA). Then, qRT‐PCR was performed with the SYBR Green Supermix Taq Kit (Takara Biotechnology Co., Ltd., Dalian, China) and analysed on an iQ5 Real‐Time PCR Detection System (Bio‐Rad Laboratories, Inc, Hercules, CA, USA). ∆∆Ct method was used to comparatively quantify the abundance of mRNA levels. The qRT‐PCR primer sequences were listed in Table 3 . The protein abundance of OXTR in placental vessels was assessed by Western blot analyses normalized to β‐actin. Antibodies were purchased from Sigma‐Aldrich or CST (Cell Signaling Technology, Inc China). All experiments were repeated three times with independently prepared tissue, and performed as previously described.19
4
Quantifying Gene and Protein Expression in Placental Vessels
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Total RNA was isolated from human placental vessels (HPV) using Trizol reagent (Invitrogen) according to manufacturer's instructions, and was then reversed transcribed using the first-strand cDNA Synthesis Kit (Toyobo Corp., Shanghai, China). qRT-PCR was performed using SYBR Green Supermix Taq Kit (Takara Biotechnology Co., Ltd., Dalian, China) and analyzed on an iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primer sequences were listed in Supplementary Table S2. ∆∆Ct method was used to comparatively quantify the amount of mRNA levels. The protein abundance of AVPR1A, AVPR2, and PKC (α, and β) in HPV was measured with Western blot normalized to β-actin. The primary antibodies were the rabbit polyclonal antibody (Santa Cruz Biotechnology) against AVPR1A, AVPR2, PKCα, PKCβ and β-actin (all 1:1000). The secondary antibody was the goat anti-rabbit antibody (1:1000; Beyotime Biotechnology, Jiangsu, China). Immuno-signals were visualized using UVP imaging system (Tianneng, Shanghai, China). Imaging signals were calculated and analyzed, and then the ratio of band brightness to β-actin was acquired to measure the relative protein expression level. Analyses was performed as previously described [25 (link),26 (link)].
5
Quantitative Analysis of HUVEC RNA and Proteins
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Total RNA from human umbilical vein endothelial cells (HUVECs) was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and was then reversed transcribed using the first-strand cDNA Synthesis Kit (Toyobo (Shanghai) Biotech, Shanghai, China). Real-time quantitative PCR was performed using the SYBR Green Supermix Taq Kit (Takara Biotechnology Co., Ltd., Dalian, China) and was analyzed using the Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primer sequences are listed in Table 2. Whole-cell proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and were immunoblotted with primary antibodies against eNOS (rabbit polyclonal, 1:200 dilution), SK3 (rabbit polyclonal, 1:200 dilution), KCNMA1 (rabbit polyclonal, 1:200 dilution), KCNMB1 (mouse monoclonal, 1:500 dilution) or NOX2 (mouse monoclonal, 1:1000 dilution) followed by incubation with the appropriate secondary antibodies. All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All experiments were repeated three times with independently prepared tissue lysates. Real-time quantitative PCR and western blot analyses were performed as previously described. 30
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