Cells cultured in 12-well plates were harvested for RNA extraction using Trizol reagent (Invitrogen, Carlsbad, United States). cDNA was synthesized from total RNA (1 μg) using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Amplification was conducted using a SYBR-Green qPCR Master Mix Kit (Vazyme, Nanjing, China) on a LC480 Lightcycler system (Roche, Indianapolis, United States). Primer pairs used in qPCR analysis were shown in Supplementary Table 1 . The expression of target genes relative to the housekeeping gene, GAPDH, was analyzed using the 2−ΔΔCt method.
Sybr green qpcr master mix kit
Manufactured by Vazyme
Sourced in China
The SYBR-Green qPCR Master Mix Kit is a ready-to-use solution for real-time quantitative PCR (qPCR) experiments, utilizing the SYBR-Green fluorescent dye technology. The kit contains all the necessary components, including a high-performance DNA polymerase, reaction buffer, and SYBR-Green dye, optimized for sensitive and reliable qPCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Lc480 lightcycler system, by Roche (1 mentions)
Prism 9, by GraphPad (1 mentions)
Hiscript 2 q select rt supermix for qpcr kit, by Vazyme (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Hiscript 2 q select rt supermix for qpcr, by Vazyme (1 mentions)
Hiscript 2 first strand cdna synthesis kit, by Vazyme (1 mentions)
Rna isolator, by Vazyme (1 mentions)
Horseradish peroxidase labeled goat anti rabbit igg, by Beyotime (1 mentions)
Anti gper antibody, by Abcam (1 mentions)
Horseradish peroxidase labeled goat antimouse igg, by Beyotime (1 mentions)
Estrogen, by Merck Group (1 mentions)
Gper inhibitor g 15, by Bio-Techne (1 mentions)
5 protocols using sybr green qpcr master mix kit
1
Gene Expression Analysis by qPCR
2
Gene Expression Analysis of Methyl Jasmonate-Treated Zinnia
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First, 0.1% Methyl Jasmonate (MeJA) was evenly sprayed onto the petals of Z. candida with consistent growth; 0.1% ethanol was used as the control treatment. Samples were collected at 2 and 4 h after treatment. RNA was extracted from the MeJA-treated and control samples using an RNA extraction kit and then reverse transcribed to cDNA using the HiScript II Q Select RT SuperMix for qPCR kit (Vazyme, Nanjing, China). Gene expression was quantified through the use of the SYBR Green qPCR Master Mix kit (Vazyme, Nanjing, China) and subsequent data analysis was accomplished through a relative quantitative approach and graphing utilizing GraphPad Prism 9 (https://www.graphpad.com/ . 1 November 2023).
3
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated from cortical and hippocampal tissues using TRIzol reagent in accordance with the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Total RNA concentrations and purity were measured using a Nanodrop 2000, and cDNA was synthesized using HiScript II Q Select RT SuperMix for qPCR (Vazyme Biotech, Nanjing, China) following the manufacturer’s instructions. RNA (600 ng) was used to synthesize cDNA in a total volume of 20 μL. cDNA template (1 μL) was employed for real-time PCR using the SYBR Green qPCR Master Mix Kit (Vazyme Biotech) with the StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Gapdh was used as a reference gene. Each sample was evaluated in triplicate or in duplicate, and gene expression was expressed as fold changes by the comparative Ct method (2−ΔΔCt). The primer sequences are shown in Supplementary Table 1 .
4
Quantifying TLR4 mRNA Expression using RT-qPCR
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Following transfection for 48 h, cells were collected and washed with PBS twice. Next, based on the one-step method (20 (link)), total RNA was extracted by adding 1 ml RNA isolator (Vazyme Biotech Co., Ltd.). As the template for PCR, cDNA was synthesized using the HiScript II First Strand cDNA Synthesis kit according to the manufacturer's protocol (Vazyme Biotech Co., Ltd.). The internal reference was human GAPDH. The fluorescent signal was obtained using SYBR-Green I. Based on the manufacturer's protocol of the qPCR SYBR-Green Master Mix kit (Vazyme Biotech Co., Ltd.), qPCR was performed. The thermocycling conditions were as follows: 95°C for 5 min (pre-denaturation), followed by 40 cycles at 95°C for 10 sec and 60°C for 30 sec, and dissociation at 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. For each sample, each experiment was repeated three times. The forward primer for GAPDH was 3′-CACTACCGTACCTGACACCA-5′ and the reverse primer was 3′-ATGTCGTTGTCCCACCACCT-5′. The TLR4 sequence was synthesized by GeneCopoeia, Inc. (cat. no. HQP054754). The relative expression levels of TLR4 mRNA (ΔCq=CqTLR4-CqGAPDH) were calculated using the 2−ΔΔCq method (21 (link)). RT-qPCR was used to evaluate the silencing effect of shRNA TLR4 expression levels and to select the interference plasmid with the greatest silencing effect.
5
Signaling Pathway Analysis of GPER in Cells
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Dulbecco's modified Eagle medium (DMEM) (Hyclone, USA), fetal bovine serum (FBS) (Hyclone, USA), estrogen (Sigma-Aldrich, USA), GPER inhibitor G15 (Tocris, USA), primary antibodies: anti-GPER antibody (Abcam, USA) and anti-AKT/phosphorylated (p)-AKT antibody (Abcam, USA), secondary antibodies: IFKine donkey anti-rabbit immunoglobulin G (IgG) fluorescence secondary antibody (Abbkine, USA), IFKine donkey antigoat IgG (Abbkine, USA), horseradish peroxidase-labeled goat anti-rabbit IgG (Beyotime, Hangzhou), horseradish peroxidase-labeled donkey anti-goat IgG (Beyotime, Hangzhou), horseradish peroxidase-labeled goat antimouse IgG (Beyotime, Hangzhou), AceQ quantitative polymerase chain reaction (qPCR) SYBR Green Master Mix Kit (Vazyme, Nanjing), HiScript II Q RT SμperMix for qPCR (+gDNA wiper) kit (Vazyme, Nanjing), optical microscope (Leica DMI 4000B/DFC425C, Germany), fluorescence qPCR instrument (ABI 7500, USA), and Image-Pro image analysis system (BIORADHERCULES, USA).
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