Talent qpcr premix

Total RNAs were isolated using Trizol (Solarbio, Beijing, China) and underwent reverse transcription to cDNA using the Promega PrimeScript RT reagent kit (Promega, Madison, WI, USA). Reverse transcription of mRNA and miRNA was performed as previously described [38 (link)]. qPCR analysis was performed on the synthesized cDNA using Talent qPCR PreMix (SYBR Green) (Tiangen, Beijing, China) with the appropriate amplification conditions: 95 °C, 3 min; 95 °C, 5 s; 50–60 °C, 10 s; 72 °C, 15 s; for 40 cycles. The qPCR primers (Table 1) were designed using Primer Premier 5 (Premier Biosoft, CA, USA) and synthesized by Sangon Biotech (Shanghai, China). The 2^−ΔΔCt method was used to quantify the expression levels and the results were normalized relative to β-actin (for mRNA) and U6 (for miRNA). Each sample was repeated three times.

Following manufacturer’s protocal, total RNA was separated from cells by Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription for RNA was performed by Fastking gDNA Dispelling RT SuperMix (Tian Gen biotech, China). Quantitative real-time RT-PCR was conducted for exploring gene expression variations with Talent qPCR PreMix (SYBR Green) (Tian Gen biotech, China). The relative expression levels were measured by adopting the 2-∆∆CT method and the mRNA expression was normalized to β-Actin expression. PCR primers can be observed from Table 3.Table 3

Primer sequences for qRT-PCR.

Gene	Forward primer	size (bp)	Reverse primer	size (bp)
Nrf2	TTCCCGGTCACATCGAGAG	18	TCCTGTTGCATACCGTCTAAATC	19
RFC4	TTGGGCCTGAACTTTTCCGAT	21	AGCGACTTCCTGACACAGTTA	21
RFC5	ACTCCTGAACTCATGGTTCCC	21	CCCTACGCATGTCTCCACT	19
PCNA	CCTGCTGGGATATTAGCTCCA	21	CAGCGGTAGGTGTCGAAGC	19
RPA1	CGGGAATGGGTTCTACTGTTTC	22	CGAGCACAAATGGTCCACTTG	21
RPA2	GCACCTTCTCAAGCCGAAAAG	21	CCCCACAATAGTGACCTGTGAAA	23
β-Actin	CTACCTCATGAAGATCCTCACCGA	24	TTCTCCTTAATGTCACGCACGATT	24

Each sample of total RNA was isolated from five normally developed terminal oocytes or resorption body 1 oocytes per female adult respectively as one biological replicate using TRIzol reagent (Invitrogen). For mRNA quantification, Oligo (dT)-primed cDNA was reverse transcribed from total RNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega). qRT-PCR was carried out using a LightCycler 480 instrument (Roche) and Talent qPCR PreMix (SYBR Green) (Tiangen) according to the manufacturer’s instructions. For miRNA quantification, the miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen) was used to synthesize the cDNA. The cDNA of miRNA was subjected to qRT-PCR using the miRcute Plus miRNA qPCR Detection Kit (SYBR Green) (Tiangen) according to the manufacturer’s instructions on a LightCycler 480 instrument (Roche). The PCR data were analyzed by the 2^-△△Ct method of relative quantification with the internal control rp49 and U6 for mRNA and miRNA, respectively. All the primers for qRT-PCR are listed in S1 Table.

Total RNA extraction from renal tissue specimens and NRK-52E cells was carried out with TRIzol reagent (Invitrogen, U.S.A.). A RevertAid™ First Strand cDNA Synthesis Kit (Thermo, U.S.A.) was employed for cDNA production. Subsequently, Talent qPCR PreMix (SYBR Green) (Tiangen, Beijing, China) was employed for qPCR. The corresponding primer sequence is shown in Table 1. Reverse transcription and quantitative detection of miR-27a were performed based on the protocols included in the RevertAid™ First Strand cDNA Synthesis Kit and Bulge-Loop™ miRNA qRT-PCR Primer Kit (RiboBio, Guangzhou, China), respectively. Relative quantification was carried out with the 2^−ΔΔCt method; U6 or β-actin served as an internal control.

Whole RNA was isolated utilizing TransZol Up Plus RNA Kit (TransGen BioTech, China). qRT-PCR was conducted using Talent qPCR PreMix (TIANGEN, China) on a ABI7900 (Thermo Fisher Scientific) system as manual instructed. The primers (forward and reverse) specific for genes were listed below:
CCNB2, 5′-CCGACGGTGTCCAGTGATTT-3′ and 5′-TGTTGTTTTGGTGGGTTGAACT-3′CCNB1, 5′- CATGGTGCACTTTCCTCCTT-3′ and 5′-AGGTAATGTTGTAGAGTTGGTGTCC-3′PLK1,5′-CACAGTGTCAATGCCTCCA-3′ and 5′-TTGCTGACCCAGAAGATGG-3′
TTK, 5′-GTGGAGCAGTACCACTAGAAATG-3 and 5′-CCCAAGTGAACCGGAAAATGA 3′;
CDC20, 5′-GCACAGTTCGCGTTCGAGA-3′ and 5′-CTGGATTTGCCAGGAGTTCGG-3′BUB1, 5′-GGAGAACGCTCTGTCAGCA-3′ and 5′-TCCAAAAACTCTTCAGCATGAG-3′PTTG1, 5′-GCCTCTCATGATCCTTGACG-3 and 5′-GCTTGAAGGAGACTGCAACA-3′
CDC45, 5′-GAAGCGCACACGGTTAGAA-3′ and 5′-GTTCACTCCCAGAGCCACTC-3′BUB1B,5′-CAGTCAGACTCTCAGCATCAAGA-3′ and 5′-CGAGGCAGAAGAACCAGAGA-3′
CDC25C, 5′-TGGGCAAATTTCTTGGTGA-3′ and 5′-AAGATCGAGGCAACGTTTTG-3′CCNA2, 5′-GGTACTGAAGTCCGGGAACC-3′ and 5′-GAAGATCCTTAAGGGGTGCAA-3′AURKA, 5′-CGCCCTGTAGGATACTGCTT-3′ and 5′-CAAATATCCCCGCACTCTG 3′
Actin, 5′-GAGCTACGAGCTGCCTGACG-3′ and 5′-CCTAGAAGCATTTGCGGTGG-3.
Actin was selected as internal reference. All the data was calculated by 2^-ΔΔt method.