Total RNA was isolated from the cumulus cells of the patients with a RNeasy Plus Micro Kit (74034; Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. Total RNA was extracted from primary cultured mouse ovarian GCs and COCs and from mouse ovary-derived GCs and COCs with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The total RNA (1 μg) was reverse-transcribed with HiScript II Reverse Transcriptase (R201-1; Vazyme, Nanjing, China). Real-time quantitative PCR (qPCR) was performed with Bestar SybrGreen qPCR Mastermix (DBI Bioscience, Germany) on an Applied Biosystems 7500 Real-time qPCR System (Applied Biosystems, Foster City, CA, USA). The relative gene expression levels were compared with those in the controls, and all real-time qPCRs were performed in triplicate. The primers are shown in Supplementary Table SI .
Applied biosystems 7500 real time qpcr system
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Applied Biosystems 7500 Real-time qPCR System is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qPCR) analysis. The system utilizes fluorescence detection technology to monitor the amplification of target DNA sequences in real-time during the PCR process.
Automatically generated - may contain errors
2 protocols using applied biosystems 7500 real time qpcr system
1
Total RNA Isolation and qPCR Analysis of Ovarian Cells
2
Quantification of IDO1 mRNA Expression
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To analyze the amount of IDO1 mRNA, real-time PCR was performed by the Applied biosystems 7500 real-time qPCR system (Applied Biosystems, Foster City, USA)using the EvaGreen PCR Master Mix (BioRad, Tokyo, Japan). The mRNA level of IDO was normalized to that for β-actin. Primers used in real-time PCR were as follows-IDO1:5′-GCATTTTTCAGTGTTCTTCGCATA-3′ (sense) and 5′-CATACACCAGACCGTCTGATAGCT-3′ (antisense); β-actin:5′-TGCACCACACCTTCTACAATGA-3′ (sense) and 5′-CAGCCTGGATAGCAACGTACAT-3′ (antisense).
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