Ncode vilo mirna cdna synthesis

Total RNA was extracted from the cells using the TRIzol reagent (15596018, Thermo Fisher Scientific, USA), and the concentration of the RNA was detected by using Nanodrop (FSC-6539918, eGeneralMedical.com, USA) and diluted to 500 ng/μL. For miRNAs, NCode VILO miRNA cDNA Synthesis and EXPRESS SYBR GreenER miRNA qRT-PCR Kits (Life Technologies) were applied for detecting the expression levels. For mRNAs, total RNA (1 μg) was converted into cDNAs using a Superscript II first-strand cDNA synthesis system (Invitrogen, USA). The mRNA expression levels were determined by SYBR-Green PCR Master Mix (Thermo Fisher Scientific, USA) in the 7500 Real-Time PCR system (Thermo Fisher Scientific, USA). Conditions of the PCR cycle were set as follows: pretreatment at 95°C for 30 s, followed by 60°C for 30 s and 60°C for 30 s for 45 cycles. The 2^−ΔΔCT method was used to determine the expression levels of RT-PCR products (Livak and Schmittgen [24 (link)]). All primer sequences used are listed in Table 1.

Total RNA was isolated from the cell lines using TRIzol (Life Technologies) and quantified. Equal amount of RNA was used for the one-step or two-step qPCR performed using the Superscript III SYBR Green qRT-PCR kits, according to manufacturer’s instructions (Life Technologies). For miRNA, PCR was performed using NCode VILO miRNA cDNA Synthesis and EXPRESS SYBR GreenER miRNA qRT-PCR Kits (Life Technologies), according to the manufacturer’s protocol. The primers (sequences provided in the Supplementary materials and methods; Additional file 7) were designed using Primer 3 [48 (link)] and synthesized by Integrated DNA Technologies (Coralville, IA). PCR was performed using Realplex² Mastercycler ep gradient S thermal cycler (Eppendorf).