Abi sequence detection system

At the end of culture, we pooled 18 to 30 follicles for each experimental condition, which were immediately flash frozen in liquid nitrogen. RNA was purified from the follicles using the RNeasy Micro Kit (Qiagen, Manchester, UK). RNA quality and quantity were assessed employing NanoDrop technology (ND-1000; Thermo Fisher Scientific) and High Sensitivity R6K ScreenTape System (Agilent, Cheshire, UK). RNA was diluted to a concentration of 50 to 100 ng/μL. RNA was reverse transcribed to complementary DNA (cDNA) using an AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Agilent Technologies) according to the instructions of the manufacturer. Messenger RNA (mRNA) expression levels were assessed by quantitative polymerase chain reaction using an ABI sequence detection system (Perkin-Elmer Applied Biosystems, Warrington, UK). All analyses were assessed in 10-μL final volume in reaction buffer, containing 2 X Taqman Universal PCR Master Mix (5.0 μL; Thermo Fisher Scientific), probe-primer mix for the target gene (0.5 μL), and 4.5 μL cDNA (100 ng) (39 (link)). All reactions were normalized against the housekeeping genes 18S ribosomal RNA and ribosomal protein L18 (Rpl18) ribosomal RNA. Data were expressed as Δcycle threshold (CT) values [ΔCT = (CT of target gene) − (CT of housekeeping gene)].

RNA was isolated from virus-infected mice hearts. Total RNA was extracted using TRIzol^® reagent (ThermoFisher Scientific, Cambridge, MA, USA) according to the manufacturer’s instructions. For RNA quantification, we synthesized complementary DNA (cDNA) using 1 µg RNA through a reverse transcription reaction using an oligo-dT primer. Real-time PCR quantitative RNA and DNA analyses were performed with an ABI Sequence Detection System using the SYBR green fluorescence quantification system (Applied Biosystems, Waltham, MA, USA). The standard PCR conditions were 95 °C for 10 min, then 40 cycles at 95 °C (30 s), and 60 °C (60 s), followed by a standard denaturation curve. All samples were tested in duplicate. The primer sequences are provided in Table 1.

For profiling the mRNA expressions of PC cells, a TRIzol kit purchased from Invitrogen (CA, USA) was used to extract total RNAs. After purification and quantification, 50 ng RNA was reverse-transcribed into a first-stand cDNA in line with the protocol of the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA). Then, on an ABI Sequence Detection System (7500, Applied Biosystems, Foster City, USA), qPCR was performed. To calculated the final expression levels (relative) of target genes, we performed the 2^−ΔΔCt method and used β-actin as the reference gene. A plasmid containing the sequence of BIRC5 was set as a positive control to monitor whether the reaction system of RT-qPCR was normal.