Quantifying lncRNA Expression via qRT-PCR

RNA extraction and qRT-PCR analysis were carried out as described in our previous study (Zhang et al., 2021a (link)). Total RNA from cultured cells was extracted using SparkZol reagent (SparkJade, Shandong, China), and cDNA was synthesized with the Evo M-MLV RT Premix for qPCR (AG11706, Accurate Biotechnology, Hunan, China). The qRT-PCR analysis was carried out using the SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biotechnology, Hunan, China) as directed by the manufacturer. Primers were synthesized by BioSune Co., Ltd. (Shanghai, China) and the sequences are shown in Table 1. The qRT-PCR was performed to evaluate the expression levels of the four lncRNAs (CTB-4E7.1, RP11-553A10.1, RP11-24N18.1, and PVRL3-AS1) using a LightCycler480 system (Roche Diagnostics, Switzerland). The lncRNAs expression was normalized to ACTB, and the relative expression level was determined by the 2^-∆∆CT method.
The study design and workflow of the present research is shown in Figure 1A. This study was approved by the Ethics Committee of Shandong Provincial Hospital (the ethics approval number is SZZJJ:NO.2021-419) and was carried out in accordance with relevant guidelines and regulations.