qPCR Analysis of mRNA and miRNA Expression

At the endpoint, the transfected cells were subjected to total RNA isolation by using NucleoZOL Reagent (Takara Bio USA, Mountain View, CA) according to the manufacturer’s introduction. For qPCR analysis of mRNA transcripts, total RNA was used for reverse transcription with the hexamer and M-MuLV (NEB). The cDNA products were diluted as templates for qPCR. The qPCR primers were designed by Primer3 Plus program [48 (link)]. For assessing the miR expression levels mediated by the three expression systems, the reverse transcription reactions were carried out by using miR-specific reverse primers that were complementary with the 3′-end six nucleotides of mature miR-5p and/or miR-3p, preceded with a 44-nt artificial stem-loop sequence. SYBR green-based quantitative real-time PCR analysis was performed by following our previously optimized TqPCR protocol [49 (link)]. The qPCR reactions were done in triplicate. All expression values were normalized to the reference gene GAPDH expression by using the 2^–ΔΔCt method [50 (link)–53 (link)]. The sequences of the qPCR primers are listed in Supplemental Table 1.