Primerscript rt master kit

TRIzol was used to obtain the total RNA, which was refrigerated at − 80 °C. The biological spectrometer was then used to assess the RNA concentration. For another, the PrimerScript RT Master kit (Takara Biotechnology, Dalian, China) was used to do cDNA synthesis. The mRNA level of MNAT1 was detected by qRT-PCR with SYBRGreen PCR Master mix (Roche, Mannheim, Germany) on an ABI 7900 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The relative fold-change in expression compared with control sample GAPDH was calculated using 2-^ΔΔCt method. The specific primers used in this study were listed as following: MNAT1 (amplicon size: 160 bp, Tm: 60 °C), 5′-GGTTGCCCTCGGTGTAAGAC-3′ (forward) and 5′-AGTTGCTCTTTCTGAGTGGAGT-3′ (reverse); GAPDH (amplicon size: 231 bp, Tm: 60 °C), 5′- GAGAAGGCTGGGGCTCATTT − 3′ (forward) and 5′- AGTGATGGCATGGACTGTGG − 3′ (reverse);

Cells were lysed in RNAiso plus (Takara, cat#D9108A) and RNA isolation was performed following manufacturer’s instructions. Extracted RNA was reverse transcribed into cDNA using the Primerscript RT master kit (Takara, cat#RR036A). cDNA samples and primers were prepared with SYBR Green Mixture (Roche, cat#04913914001) and detected using Applied Biosystems Stepone Plus detection system. The results were processed by ΔΔCt algorithm. Primers used in qPCR analysis were as followed (5′-3′):
Mouse Gapdh-F, TGTGATGGGTGTGAACCACGAGAA.
Mouse Gapdh-R, CTGTGGTCATGAGCCCTTCCACAA.
Mouse Adam17-F, ACCACTTTGGTGCCTTTCGT.
Mouse Adam17-R, GTCGCAGACTGTAGATCCCTT.
Mouse Procr-F, GCATGTTGACGAAGTTTCTGCCG.
Mouse Procr-R, GCTTAGCAACGCCGTCCACTTG.
Human GAPDH-F, ACATCGCTCAGACACCATG.
Human GAPDH-R, TGTAGTTGAGGTCAATGAAGGG.
Human ADAM17-F, GTGGATGGTAAAAACGAAAGCG.
Human ADAM17-R, GGCTAGAACCCTAGAGTCAGG.
Human PROCR-F, GCATGTTGACAACATTGCTGCCG.
Human PROCR-R, GCTTAACATCGCCGTCCACCTG.

TRIzol regent (Thermo Fisher Scientific) was utilized to extract total RNA, which was preserved at −80°C. RNA concentrations were assessed using a biological spectrometer. cDNA synthesis was carried out using the PrimerScript RT Master kit from Takara Biotechnology (Dalian, China). FOXD2‐AS1, miR‐206, and MAP3K1 levels were quantified using RT‐qPCR by employing SYBR Green PCR Master mix (Roche, Mannheim, Germany) and an Applied Biosystems 7900 Real‐Time PCR System. Changes in FOXD2‐AS1 levels in the serum were calculated using 2^−ΔCt. Relative gene expression was calculated using 2^−ΔΔCt. The related primer sequences were shown in Table 1.