Cfx96 pcr system

RT-qPCR was performed to validate the RNA-Seq results for lncRNA expression. Total RNA was extracted from 100 fertilized embryos using the Qiagen RNeasy Mini kit (Qiagen, Inc.), according to the manufacturer's protocol. RNA then underwent RT using the PrimeScript™ RT Reagent kit with gDNA Eraser (perfect real time; Takara Bio, Inc., Otsu, Japan). The relative expression levels of the target lncRNAs were measured by RT-qPCR using the Power SYBR-Green RT-PCR kit (Toyobo Life Science, Osaka, Japan) in a Bio-Rad CFX96 PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). H2A histone family member Z was employed as a housekeeping gene, and each sample was analysed three times. The lncRNA-specific PCR conditions were as follows: Initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and extension at 72°C for 30 sec. RT-qPCR primers used in these analyses are presented in Table I.
The comparative quantification cycle (Cq) method was employed to quantify the relative expression of lncRNAs. The expression levels of the lncRNAs were analysed as FC values using the ΔCq method (27 (link)). All data were log transformed, and the results were comparable to the RNA-Seq data.

Total RNA from tissues and cells were extracted using the Trizol reagent (Invitrogen, USA). Reverse transcription was performed using the PrimeScript™ RT reagent with the gDNA Eraser Kit (TaKaRa, Japan) according to the manufacturer’s instructions. The cDNA samples were detected using TB Green^® Premix Ex Taq™ II (Tli RNaseH Plus, TaKaRa, Japan) and the Bio-Rad CFX96 PCR System (Bio-Rad, Hercules, CA, USA) with CFX Manager Software (version 3.1, Bio-Rad). The primer sequences were: PINK1 forward primer: 5ʹ-CAAGAGAGGTCCCAAGCAAC-3ʹ, PINK1 reverse primer: 5ʹ-GGCAGCACATCAGGGTAGTC-3ʹ; GAPDH forward primer: 5ʹ-GGACCTGACCTGCCGTCTAG-3ʹ, GADPH reverse primer: 5ʹ-CCTGCTTCACCACCTTCTTGA-3ʹ.

Total RNA was extracted from cells and tissues with TRNzol Universal Reagent (TIANGEN BIOTECH, Beijing, China) following the manufacturer’s protocol. The qRT-PCR reaction was performed with Quant One Step qRT-PCR Kit (Probe, TIANGEN BIOTECH, Beijing, China) by using the Bio-Rad CFX96 PCR System (Bio-Rad, CA, USA). The thermocycling conditions were as follows: A holding step at 95°C for 30 sec, and 40 cycles at 95°C for 5 sec and 60°C for 30 sec. All primers were designed as follows: GSKIP, forward 5ʹ -CATGGTGGTACCTGCTTGTG-3ʹ, reverse 5ʹ -GAAAGGGTCTTGCTCTGTGG-3ʹ; miR-181c-5p, forward 5ʹ -UUGUAAGUUGGACAGCCACUCA-3ʹ, reverse 5ʹ -TGGTGTCGTGGAGTCG-3ʹ, GAPDH, forward 5ʹ-TAGCTGAACCGTAACTGGC-3ʹ, reverse 5ʹ-CCTAGGAACTAACGGCGTT-3ʹ. CT value was normalized to GAPDH and calculated with the 2^−ΔΔCt.

Total RNA was extracted with TRIzol (Invitrogen). Cytoplasmic and nuclear RNA were extracted with PARISTM Kit (Invitrogen). PrimeScript RT Master Mix and SYBR Premix Ex Taq II (Takara, Japan) were used to conduct qRT-PCR with Bio-Rad CFX96 PCR System (USA). The primers were synthesized by Invitrogen (Table S1). For the miRNA, All in One miRNA qRT-PCR Detection Kit (GeneCopoeia) was used. Threshold cycle (CT) value was normalized to β-actin or U6 with the 2^−ΔΔCt method.