480 qpcr system

Total RNA from tissues and cells was extracted with TRIzol reagent (Takara, Otsu, Japan) according to the manufacturer's instructions.¹⁰ The concentration and purity of RNA were measured using a NanoDrop ONE spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The reverse transcription of WEE1 and lncRNA XIST was performed using the High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The transcription of microRNA was performed by the All‐in‐One First‐Strand Synthesis Kit (GeneCopoeia, Rockville, Montgomery, USA). A total of 2 μg RNA from each sample was transcribed for PCR detection. Total RNA was transcribed by a reverse transcription kit purchased from Takara. Real‐time PCR was carried out with SYBR Green Real‐Time PCR Master Mix (Toyobo, Osaka, Japan) using GAPDH as the reference. Real‐time PCR for microRNA was carried out by the All‐in‐One miRNA qRT‐PCR Detection Kit (GeneCopoeia, Rockville, Montgomery, USA) with U48 as a reference for microRNA. Real‐time qPCR was performed with the Roche 480 qPCR System (Roche Diagnostics, Basel, Switzerland), and fold changes were calculated according to the relative quantification 2^−∆CT method.

The expression levels of lncRNA EPB41L4A-AS1, KB-1732A1.1, RP11-390P2.4, RP11-421L21.3 and HOTAIR were assessed by quantitative real-time PCR (qRT-PCR). The oligonucleotide primers used for real-time PCR are listed in Table 1. Total RNA was extracted from 12 paired samples (12 NSCLC tissues and 12 adjacent non-cancerous tissues) using TRIzol^TM Reagent (Invitrogen^TM) according to the manufacturer’s instructions. Then, total RNA was reversed to cDNA using a PrimeScript^TM RT Reagent kit (TaKaRa) and cDNAs were quantified by real-time PCR amplification using a SYBR^® Premix Ex TaqTM kit (TaKaRa) on Roche 480 qPCR System (Roche Life Science). The relative expression level was determined with the 2^–ΔΔCt method and GAPDH was assessed as an internal control. Each sample was performed in triplicate.