Mircute plus mirna qpcr kit

The total RNA in VECs was extracted using TRIzol reagent (Invitrogen, USA) following the manufacturer's instructions. After RNA extraction, all RNA samples were reverse transcribed using the first‐strand cDNA Synthesis Kit (Takara, Dalian, China). Then, quantitative polymerase chain reaction (PCR) was conducted using the iQ5 Real‐Time PCR Detection System (Bio‐Rad) with SYBR Green PCR Master Mix (Applied Biosystems, USA) for mRNA analysis. The relative mRNA levels of genes were calculated using the ∆∆Ct method with GAPDH as the normalization control. For miRNA analysis, miRNAs from VECs were extracted using the miRcute miRNA isolation kit (Tiangen, Beijing, China). The miRcute plus miRNA first‐strand cDNA kit (Tiangen, Beijing, China) was used for miRNA reverse transcription. Quantitative PCR of miRNAs was performed to determine their relative levels using the miRcute plus miRNA qPCR kit (SYBR Green) and analyzed on the iQ5 Real‐Time PCR Detection System (Bio‐Rad). The expression of U6 was used as the normalization control for each miRNA sample. The primers used in this study are listed in Table. All experiments were repeated 3 times using independently prepared cells and were performed as previously described.
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