The cultured cells were treated with metformin (0–0.75 mM) for 48 h. Total cell lysate was collected and mRNA was extracted using the RNeasy kit (QIAGEN, Venlo, The Netherlands). cDNA was then synthesized from 1.5 μg of total mRNA using reverse transcriptase (Invitrogen, Carlsbad, CA, USA), as per the manufacturer’s instructions. Real-time quantitative PCR was performed by combining 2.5 μL of the RT with 22.5 μL of SYBR green (Roche, Basel, Switzerland). The reaction was run using System 7300 (Applied Biosystems, Foster City, CA, USA) and results were analyzed by the relative quantity method. Experiments were performed in triplicates with GAPDH expression as the endogenous control. siRNA for the Rieske protein (UQCRFS1) was obtained from Dharmacon, Lafayette, CO, USA. Primers were custom designed by the authors and created by Eurofin Genomics, Louisville, KY, USA. The following sequences were used:
System 7300
Manufactured by Thermo Fisher Scientific
Sourced in United States
The System 7300 is a high-performance laboratory instrument designed for analytical applications. It features advanced technology and precision engineering to ensure reliable and accurate results. The core function of the System 7300 is to provide precise and efficient analysis of samples across a range of scientific disciplines.
Automatically generated - may contain errors
4 protocols using system 7300
1
Metformin Transcriptional Regulation Assay
2
RNA Isolation and qPCR Analysis
3
Gene Expression Analysis of uPA and HPRT-1
4
RNA Isolation and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total RNA was isolated using the RNeasy Kit (Qiagen). cDNA was synthesized with the iScript cDNA Synthesis Kit (Bio-Rad). qPCR was performed on a System 7300 instrument (Applied Biosystems) with a one-step program: 95°C for 10 min, 95°C for 30 s, and 60°C for 1 min for 40 cycles. HPRT-1 gene expression was measured as a normalizer. Results were analyzed by the relative quantity method. Experiments were performed in triplicate with internal duplicate determinations.
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