Cfx connect real time pcr detection instrument

According to the manufacturer’s instructions, total RNA was extracted from whole ovaries by TRIzol reagent (Invitrogen, United States) and miRNA or mRNA easy Minikit (Qiagen, Valencia, CA, United States). In addition, RNA concentrations were determined by NanoDrop ND-1000 spectrophotometer (Wilmington, DE, United States).
For qRT-PCR experiments, the RNA was reverse-transcribed into cDNA with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, United States) following the manufacturer’s instructions. Quantitative PCR reaction was run on a CFX Connect Real-Time PCR Detection Instrument (BioRad, United States). Expression of the selected miRNAs was quantified by qRT-PCR, after reverse transcription with miRNA-specific stem-loop primers. The primer sequences are shown in Tables 1, 2 to amplify fragments. The data were normalized to expression levels of the housekeeping genes U6 and GAPDH, respectively, and the 2^−ΔΔCT method was used to calculate relative expression levels.

Total RNA was extracted using FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme) and reverse transcription with a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme) following the manufacturer’s instructions. qPCR was performed using SYBR Green Master Mix (Vazyme) on a Bio-Rad CFX Connect Real-Time PCR Detection instrument. Primer sequences are listed in the Table S6. The expression of all samples was normalized to 18S rRNA.

After quantification and quality check of total RNA by electrophoresis, 1,000 ng of total RNA was reverse transcribed using SuperScript IV Reverse Transcriptase (Invitrogen™, Carlsbad, CA). Real‐time quantitative PCR was performed using iTaq Universal SYBR Green Supermix (Bio‐Rad, Hercules, CA) on CFX Connect real‐time PCR detection instrument (Bio‐Rad, Hercules, CA). For list and details of primers, refer to Table S1. 18S rRNA expression levels were used for normalization. Fold change was determined by ΔΔCt method.