Abi 7500ht real time pcr detection system

TRIzol Reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA from follicles. The cDNA was generated from 2 μg total RNA using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co. Ltd., Nanjing, China), following the manufacturer's protocol. The qRT-PCR was performed using a SYBR® Premix Ex Taq™ kit (Takara, DRR420A, Kyoto, Japan) on an ABI 7500HT Real-time PCR detection system (Applied Biosystems, Foster City, USA), with the following conditions: 95°C for 10 min and then 40 cycles of 95°C for 30 s, 64°C for 34 s, and 72°C for 30 s. Comparisons of expression levels were determined by the 2^−ΔΔCt formula method normalized to β-actin. The sequences of primers are listed in Table 1. RNA-seq was carried out according to a previous study [38 (link)].

Total RNA was extracted from ovarian tissues using TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer's protocol. The cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, San Jose, CA, USA) following the manufacturer's instruction. The reverse transcription product was diluted 1 : 10 and then used as a cDNA template for qRT-PCR analysis. The qRT-PCR was carried out in an ABI 7500HT Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with the reaction volume of 20 μL that contained 2 μL cDNA template, 400 nM of each of the gene-specific forward and reverse primers, 0.4 μL Rox reference dye II, 10 μL SYBR Premix Ex Taq (TaKaRa, Shiga, Japan), and 6.8 μL water. qRT-PCR conditions were as follows: 95°C for 10 min and then 40 cycles of 95°C for 30 s, 60°C for 34 s, and 72°C for 30 s. Comparisons of expression levels were determined by delta CT method normalized to β-actin. Sequences of the primers are provided in Table 1.