TRIzol® reagent (Thermo Fisher Scientific, Inc.) was used for the extraction of total RNA from the cells. NanoDrop ND-1000 UV/visible photometer (Thermo Fisher Scientific, Inc.) was used to assess RNA purity. Reactions were performed using the ChamQ universal SYBR master mix (Vazyme Biotech) in the Bio-Rad CFX96 RealTime System. Pre-incubation at 95°C for 120 sec, amplification at 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec and then a final extension at 72°C for 5 min. mRNA expression was calculated using the 2−ΔΔCq method (30 (link)). Shanghai Biological Engineering Technology Co. designed and synthesized all primers used (5′-3′) (Table SII ). All data were normalized relative to GAPDH.
Chamq universal sybr master mix
Manufactured by Vazyme
Sourced in China
ChamQ Universal SYBR Master Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) applications. It contains all the necessary reagents, including a high-performance DNA polymerase, SYBR Green I dye, and optimized buffer components, to facilitate efficient and sensitive detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Cfx96 real time system, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Abi steponeplus real time pcr system, by Vazyme (1 mentions)
Hiscript 3 rt supermix with gdna wiper, by Vazyme (1 mentions)
Qs6 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Gdna wiper, by Vazyme (1 mentions)
Hiscript 2 q rt supermix for qpcr, by Vazyme (1 mentions)
Unlq 10 column total rna purification kit, by Sangon (1 mentions)
Hiscript 2 reverse transcriptase, by Vazyme (1 mentions)
Total rna isolation kit, by Vazyme (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Rna easy isolation reagent, by Vazyme (1 mentions)
Hiscript 2 q rt supermix kit, by Vazyme (1 mentions)
8 protocols using chamq universal sybr master mix
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
Quantifying mRNA Expression of CSE Gene
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Total RNA was extracted from the cells with Trizol reagent and reverse transcribed with Vazyme RT kit (Nanjing, China) in accordance with a standard protocol.qRT-PCR was performed on an ABI StepOnePlus Real-Time PCR System (Vazyme,Nanjing, China) using ChamQ Universal SYBR Master Mix. The 2−△△Ct method was used to evaluate the mRNA expression levels of CSE gene. The following primers were used in the RT-qPCR assay: CSE (5′-CCCATCTCACTGTCCACCAC-3′; 5′-GTGCTGCCACTGCTTTTTCA-3′), and GAPDH (5′-GCACCGTCAAGGCTGAGAAC-3′; 5′-TGGTGAAGACGCCAGTGGA′).
3
Macrolide Resistance in Mycobacteria
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Seven homolog efflux pump genes and the regulatory gene whiB7 (MAB_3508c) correlate with macrolides resistance in mycobacteria, were selected and analyzed. Relative expression of the genes was assessed by comparing the quantity of mRNA expressed by the organism cultured in the presence and absence of CLA using the same technical approach reported previously.28 A culture incubated in the presence of half its MIC of CLA was shaken at 37°C for 3 h; the RNA was then extracted according to the protocols described by Medjahed et al29 cDNA was synthesized using the HiScript III RT SuperMix with gDNA wiper (Vazyme Biotech Co., Ltd). qRT-PCR was performed using ChamQ Universal SYBR Master Mix (Vazyme Biotech Co., Ltd) on a QS6 Real-Time PCR System (Applied Biosystems, Carlsbad, CA). SigA was chosen as the endogenous reference gene. All PCR primer pairs used for amplification are shown in Supplementary Table 1
4
Cd2+ Stress Response Analysis in Cyanobacteria
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Three genes related to Cd2+ adsorption and one gene involved in exopolysaccharides synthesis were selected to calculate expression levels by RT-qPCR. After 15 min, 24, 48, 72, and 96 h of stress with or without 1.0 mg L−1 Cd2+, the cyanobacterial suspensions were used to extract total RNA. Total RNA was isolated from the control and stress groups using a plant RNA extraction kit (Tiangen, China). Specific primers for RT-qPCR were designed using Primer Premier 5.0, synthesized by Tsingke Biological Technology (Changsha, China), and the gap1 gene was used as an internal control gene14 (link) (Table 1 ). The cDNA synthesized by HiScript II Q RT SuperMix for qPCR containing a gDNA wiper (Vazyme, China) was used as a template for RT-qPCR. RT-qPCR was carried out on a Real-Time PCR Detection Systerm (Gentier 48R, China) with ChamQ Universal SYBR Master Mix (Vazyme, China) according to the manufacturer's instructions. Three experiments were conducted to ensure the accuracy of the results. Relative expression levels were calculated using the 2−ΔΔCT method.25 (link)
5
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from the primary macrophages (AM and BMDM) using a UNlQ-10 Column Total RNA Purification Kit (Sangon Biotech, China). 50 ng total RNA was reverse-transcribed into cDNA using a HiScript II Reverse Transcriptase (Vazyme Biotech Co., China). Real-time quantitative PCR was carried out with a ChamQ Universal SYBR Master Mix (Vazyme Biotech Co., China) as per the manufacturer's instructions on StepOne & StepOnePlus Real-Time PCR Systems (Thermo Fisher Scientific, Waltham, USA). The primer sequences used in this study are shown in Table 1 . The gene expression was quantified using the threshold cycle (Ct) values by the 2−ΔΔct method, while the expression of β-actin was used as the internal control.
6
Brown Adipocytes RNA Extraction and qRT-PCR
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After 6 days of induced differentiation, brown adipocytes were collected for RNA extraction using a Total RNA Isolation Kit (Vazyme, Nanjing, China). 0.5-0.8 μg of RNA was reverse transcribed to cDNA using Maxime RT Premix (Vazyme, Nanjing, China). Then, a qRT-PCR assay was performed with the CFX96 real-time PCR detection system (Bio-Rad, USA) using ChamQ Universal SYBR Master Mix (Vazyme, Nanjing, China). Target genes were normalized to the β-actin gene, and their respective relative expression was analysed by the 2 -ΔΔCt method. The primer sequences are listed in Table 2.
7
Quantitative Real-Time PCR Analysis
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Total cellular RNA was extracted using an RNA-Quick Puri cation Kit (Yishan Biotechnology Co., Shanghai, China) and reverse transcribed to cDNA using a HiScript RT kit (Vazyme, Nanjing, China) in accordance with the manufacturer's protocol. RT-qPCR was performed using speci c primers for the quanti cation of GLUT1, HK2, GPI, GAPDH, PGK1, PKM2 and LDHA (Table 1). β-actin was used as a control. Reactions were performed using the ChamQ universal SYBR master mix (Vazyme, Nanjing, China) in the Bio-Rad CFX96 Real-Time System. mRNA expression was calculated using the 2 -ΔΔCT method.
8
Investigating Innate Immunity Pathways
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Total RNA was extracted from the jejunum tissues (every piglet) with RNA-easy TM Isolation Reagent (L/N.7E371G9, Vazyme, China), cDNA was synthesized from the extracted RNA by reverse transcription using an HiScript ® II Q RT Super Mix kit (L/N.7E421E0, Vazyme, China), and real-time RT-qPCR was performed using the Chamq Universal SYBR Master Mix (L/N.7E472E0, Vazyme, China). The levels of TLR2, TLR3, MAVS, IRF3, MDA5, IFN-α, IFN-λ 1 and NF-κb were detected by Real-Time PCR with the primers listed in Table 2. The cycling parameters were 95 °C for 10 min; 40 cycles at 94 °C for 30 sec; 60 °C for 1 min. The speci c pig gene primers were designed on NCBI and synthesized by Sangon Biotech Corporation (Wuhan, China).
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