Hairpin it microrna qpcr quantitation kit

Total RNA was extracted with TRIzol Reagent as above described. cDNA generation and real-time PCR were performed using Hairpin-it microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China). All PCR reactions were performed using standard PCR conditions. RNU6 was used as endogenous control. Data were generated using CFX Manager software (Bio-Rad, CA, USA). The results were analyzed based on the 2^−ΔΔct method.

Total RNA was isolated using the TRIzol^® reagent (Invitrogen), and cDNA was synthesized using the HiScript^® II Q Select RT SuperMix for qPCR kit (Vazyme Biotech Co., Ltd). Quantitative RT‐qPCR was performed in duplicates using FastStart Universal SYBR Green Master (Roche Applied Science) using the StepOne real‐time PCR System (Life Technologies Corp.). GAPDH was used as an endogenous normalization control to obtained relative expression data. The expression levels of miRNA were assessed using a Hairpin‐it™ microRNA qPCR Quantitation Kit (GenePharma) according to the standard protocol. The expression of U6 was used as an endogenous control. All primer sequences are shown in Table 1.

Total RNA was extracted by TRIzol (Invitrogen, USA) according to the manufacturer's recommendation and then reverse-transcribed into cDNA using the Reverse Transcriptase M-MLV (TaKaRa) and Hairpin-it™ microRNA qPCR Quantitation Kit (GenePharma, Shanghai, China). qPCR was performed using the SYBR® Select Master Mix kit and standard protocols of the Step One Plus Real-Time PCR System (Applied Biosystems, USA). U6 small nuclear RNA was used as an internal control for miRNAs, and the mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The 2^−ΔΔCt comparative method was used to analyze expression levels. The mRNA, miRNA and U6 primers are listed in Supplementary Tables 1, 2.