Stem-Loop RT-qPCR for miRNA Analysis

Since stem-loop RT primers show better efficiency and specificity compared to conventional primers (14 (link)), the RT reaction was conducted using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific) and stem-loop RT primers (Guangzhou RiboBio Co., Ltd., Guangzhou, China). Fermentas^® RNase-free DNase I (Thermo Fisher Scientific) was used to remove genomic DNA from the RNA samples. qPCR was performed with the FastStart Universal SYBR-Green Master mix (Roche Applied Science, Indianapolis, IN, USA) on a Corbett Rotor-Gene 3000 system (Qiagen, Valencia, CA, USA). Following amplification, melting-curve analysis was performed as described by the manufacturer. Each RNA sample was analyzed in triplicate, and all measurements contained a negative control with no cDNA template and an endogenous control with an rno-U6 small nuclear RNA (snRNA). The relative expression levels of the miRNAs were calculated with the comparative cycle threshold (Ct) method using the REST 2009 software (Qiagen) (15 (link)).