Quantifying mRNA and miRNA Expression

Total RNA was prepared from cells using TRIzol reagent (Ambion, Austin, TX, United States) according to the manufacturer’s instructions. RNA quality was measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). Approximately 500 ng of RNA was used for cDNA synthesis with a HiScript III RT SuperMix for qPCR kit (Vazyme, Nanjing, China). ChamQ SYBR qPCR Master Mix Kit (Vazyme) was used to quantify mRNA expression, and GAPDH was used as an internal control. The mRNA primers are described in Table 1. For the specific detection of miR-192-5p expression, the stem-loop real-time PCR was performed as previously described (Chen et al., 2005 (link)). Approximately 1 μg of RNA was used for cDNA synthesis with a miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme). The miRNA Universal SYBR qPCR Master Mix (Vazyme) was used to quantify miR-192-5p expression, and RNU6 was used as an internal control. The miRNA primers for reverse transcription and quantification are described in Table 2. PCR reactions were conducted on ABI StepOne PCR system (Applied Biosystems, Foster City, CA, United States) and results were analyzed with the ΔΔCt method.