Bulge loop mirna qrt pcr detection kit

Sixty additional individuals including 20 CAD patients, 20 CAD high-risk patients and 20 healthy controls were recruited, blood samples were drawn and total RNA was extracted using TRIzol LS and RNeasy mini kit. Total RNA from cultured cells or animal models were isolated using TRIzol (Invitrogen, Life Technologies Corporation). Two microgram of total RNA was reverse-transcribed using PrimeScript RT-PCR Kit (Takara Bio Co. Ltd., Dalian, China). qRT-PCR were performed with the Bulge-Loop™ miRNA qRT-PCR Detection Kit (Ribobio Co.) and SYBR® Premix Ex Taq™ (Takara Bio Co. Ltd.) according to the manufacturer's protocol with the Rotor-Gene 6000 system (Corbett Life Science, QIAGEN, Hilden, Germany). The U6 small RNA was used as the housekeeping small RNA reference gene. The relative gene expression was normalized to U6. Each reaction was performed in triplicate, and analysis was performed by the 2^−ΔΔCt method as described previously 24 (link).

Two microgram of total RNA was reverse-transcribed using Transcript First-strand cDNA synthesis SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer's protocol. Briefly, 50 µL reagents were incubated for 60 min at 42°C, 10 min at 70°C, and then preserved at 4°C. The Bulge-Loop miRNA qRT-PCR Detection Kit (Ribobio Co., Guangzhou, China) and SYBR Green PCR SuperMix Kit (TransGen Biotech, Beijing, China) were used in real-time PCR for examining the relative quantification of circulating miRNAs according to the manufacturer's protocol with the Rotor-Gene 6000 system (Corbett Life Science, QIAGEN, Hilden, Germany). U6 was measured as endogenous control for normalizing the date of experimental qRT-PCR. Each specimen was measured in triplicate. The threshold cycle (Ct) value was defined as the cycle number at which the fluorescence signals passed the fixed threshold. In our experiment, the detection limit of Ct value was defined as 40 [32] (link), [33] (link), [45] (link).