Mircute plus microrna sybr green qpcr kit

Total RNA in serum samples was extracted using the miRcute serum/plasma microRNA isolation kit (cat# DP503; TianGen, Beijing, China). MicroRNA155 and microRNA29 complementary deoxyribonucleic acid were synthesized from total microRNA using the miRcute Plus microRNA first strand cDNA kit (cat# KR211; TianGen). Relative gene expression was quantified using the miRcute plus microRNA SYBR Green qPCR Kit (cat# FP401; TianGen).
Real‐time quantitative polymerase chain reaction (qPCR) was carried out using an ABI7500 Fast instrument (Applied Biosystems, Shanghai, China). Relative gene expression levels were calculated as the ratio of the target gene to an internal reference (U6 transcript). Detailed thermocycling conditions can be found in Tables S1 and S2.

miR‐4431 was extracted using the miRcute miRNA isolation kit (cat# DP503; TianGen, Beijing, China). The miRcute Plus microRNA first strand cDNA kit (cat# KR211; TianGen, Beijing, China) was used for reverse transcription of miRNA first chain cDNA, and the miRcute Plus microRNA SYBR Green qPCR Kit (cat# FP401; TianGen) was used to detect the expression of microRNA. U6 was used as an internal reference to calculate the relative expression of miR‐4431. The hsa‐miR‐4431 sequence is presented in Table S1.
Total RNA was extracted from cells using TRIZOL reagent (cat# 15596‐026; Life Technologies, California, USA), and reverse transcription was performed at 42°C for 60 min and then at 70°C for 15 min. PCR amplification was performed using a qRT‐PCR instrument (Qiagen, Hilden, Germany) with the following program settings: 95°C for 3–5 min, 40–45 cycles at 95°C for 10 s, 50–60°C for 30 s, and 72°C for 40 s. GAPDH was used for standardization. The primer sequences are listed in Table S2.