The constructed plasmid pET21a-CaCDC42 containing the cdc42 gene of Candida albicans, which served as the template DNA, was recombined and prepared in the College of Life Science, Beijing Normal University, China. SYBR® Premix Ex Taq™ II containing Taq DNA polymerase catalyzing the elongation step of DNA replication, dNTPs, ROX Reference Dye II, DNA loading buffer, and SYBR Green II was purchased from Takara Bio Inc. (Dalian, China). The synthetic primers targeting cdc42 were provided by Sangon Biotech Co. Ltd. (Beijing, China) as follows: 5′-GTGATGGTGCCGTTGGTA-3′ (forward) and 5′-CCCTGTTCCTGGGTGATT-3′ (reverse), which produced a piece of DNA containing 397 bp. Thirteen heavy metal salts, ZnCl2, Pb(NO3)2, CdCl2·2.5H2O, Ni(NO3)2·6H2O, HgCl2, CrCl3, MnCl2, CuSO4, CoCl2, Ce(NO3)3, Sr(NO3)2, Bi(NO3)3 and K3Fe(CN)6 were obtained from Beijing Chemical Reagent Company (Beijing, China). Ethylene diamine tetraacetic acid (EDTA), Ethidium bromide (EB), and agarose was from Sigma-Aldrich (St. Louis, USA). The DNA replication reactions in vitro were fulfilled and monitored using the Real-time PCR System ABI7500 (Applied Biosystems™, USA).
Real time pcr system abi7500
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI7500 Real-time PCR System is a thermal cycler designed for quantitative real-time PCR analysis. It provides precise temperature control and detection capabilities for accurate and reliable gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Agarose, by Merck Group (1 mentions)
Ni no3 2 6h2o, by Beijing Chemical Works (1 mentions)
Dntps, by Takara Bio (1 mentions)
Rox reference dye 2, by Takara Bio (1 mentions)
Dna loading buffer, by Takara Bio (1 mentions)
Sybr green 2, by Takara Bio (1 mentions)
Mncl2, by Beijing Chemical Works (1 mentions)
Zncl2, by Beijing Chemical Works (1 mentions)
Pb no3 2, by Beijing Chemical Works (1 mentions)
Hgcl2, by Beijing Chemical Works (1 mentions)
Cuso4, by Beijing Chemical Works (1 mentions)
Cocl2, by Beijing Chemical Works (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Vazyme (1 mentions)
Thermo nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr kit, by Thermo Fisher Scientific (1 mentions)
Trizol solution, by Promega (1 mentions)
Sybr premix dimereraser kit, by Takara Bio (1 mentions)
5 protocols using real time pcr system abi7500
1
Quantitative PCR of Candida CDC42 Gene
2
Quantifying Gene Expression in BEAS-2B Cells
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Total RNA from cultured BEAS-2B cells was extracted by TRIzol reagents (Invitrogen, Carlsbad, CA, United States). First-strand cDNA was generated from 2 μg of total RNA with Maxime RT PreMix kit (Takara Bio, Inc.) in accordance with the manufacturer’s recommendations. qRT-PCR was conducted using a Real-Time PCR System ABI 7500 (Applied Biosystems; Thermo Fisher Scientific, Inc.). GDPDH served as an internal control for normalization. The expressions of target mRNAs were measured using the 2−∆∆cq method.
3
Spinal Cord Tissue RNA Extraction and qPCR
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The Quick-RNA™ MiniPrep kit was used to isolate the total RNA of the T9 to T11 spinal cord segments according to the manufacturer’s instructions (R223-01, Vazyme, Nanjing, China) and Thermo Nano Drop 2000 (2000, Thermo Fisher Scientific, Massachusetts, USA) was used to analyze the purity and concentration of the extracted RNA. To convert the complementary DNA (cDNA), the High-Capacity cDNA Reverse Transcription kit (Q111-02/03, Vazyme, Nanjing, China) was used to perform the reverse transcription. Primers of were designed as
The Real-Time PCR System (ABI 7500, Applied Biosystems, Massachusetts, USA) was used to perform qPCR with the cycling parameters set as denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Each reaction was repeated 3 times and the relative quantification method of ΔΔCt was used to analyze the data, shown as relative gene expression levels compared to the control.
The Real-Time PCR System (ABI 7500, Applied Biosystems, Massachusetts, USA) was used to perform qPCR with the cycling parameters set as denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Each reaction was repeated 3 times and the relative quantification method of ΔΔCt was used to analyze the data, shown as relative gene expression levels compared to the control.
4
Quantitative PCR Analysis of Gene Expression
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Total RNA was extracted from colon tissues and HT-29 cells using TRIzol reagent and then reverse-transcribed into DNA using an RT-PCR Kit (#K1622, Thermo Fisher Scientific) according to the manufacturer's instructions. Real-time PCR was performed with the SYBR Green PCR kit (F-415XL, Thermo Fisher Scientific) on a Real-Time PCR System (ABI-7500, Applied Biosystems, MA, USA) using the GAPDH gene as an internal control [19 (link)]. The 2-ΔΔCt method was used to analyze the data [20 ]. Primers used for qPCR were purchased from Sangon Biotech (Shanghai, China). Primers were designed using the Primer-BLAST tool [21 (link)].
5
RT-qPCR Gene Expression Analysis
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We extracted total RNA according to the instruction of TRIzol solution (Promega, Madison, Wisconsin, WI, USA). Then, we utilized a PrimeScript RT reagent Kit (Takara, Dalian, China) to reverse-transcribed RNA to cDNA. Using specific primers, the gene expressions were qualified by RT-qPCR based on a commercial kit, SYBR® Premix DimerEraser Kit (Takara, Dalian, China), β-actin as control. PCR program was conducted on a Real-Time PCR System (ABI7500, Applied Biosystems, Waltham, MA, USA) and the detailed process went as follows: 1 min at 95 °C, 20 s at 95 °C and 10 s at 56 °C and 15 s at 72 °C for 35 cycles, then held at 4 °C. Finally, the calculation of gene expression was achieved by using the 2−ΔΔCt method.
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