Iq sybr green supermix with opticon

Total RNA from BUMPT cells and kidneys of C57BL/6J mice was extracted using a Trizol reagent (Accurate Biology, ChangSha, China) kit [8 (link), 26 (link), 27 (link)]. Total RNA (40 ng) was reverse-transcribed using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Accurate Biology). Real-time qPCR was used to examine the expression levels of miRNA, mRNA, and lncRNA by Bio-Rad (Hercules, CA, USA) iQ SYBR Green Supermix with Opticon (Accurate Biology, ChangSha, China) per the manufacturer’s instructions. The sequences of lncRNA ENSMUST_147219 were obtained from Ensembl database and those of miR-221-5p were obtained from miRDB. We used the following primers: ENSMUST_147219: 5′-TTTCACCCGCTTT GCCAAGTCTC-3′ (forward) and 5′-ATCCCTTCCCTGTCTCCTCAACTG-3′ (reverse); miR-221-5p: 5′-GCGACCTGGCATACAATGTAGAT-3′ (forward) and 5′-AGTGCAGGGTCC GAGGTATT-3′ (reverse); IRF6: 5′-ACAAACTGCTCTTCTATGGGCTTCTG-3′ (forward) and 5′-TCCTCCTCCTCATCTTCATCCACATC-3′ (reverse); β-actin: 5′-GGCTGTATTCCCCTCCATCG-3′ (forward) and 5′-CCAGTTGGTAACAATGCCATGT-3′ (reverse); and U6: 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse). ΔCt values were used to carry out the relative quantification.