qRT-PCR Validation of lncRNAs in Plasma

The relative levels of selected lncRNA transcripts were quantified by qRT-PCR using specific primers. RNA was firstly removed from genomic DNA and reverse transcribed to cDNA using a PrimeScript^® RT reagent Kit with gDNA Eraser (Takara Code: DRR047, Japan). Then, with the diluted cDNA as a template and β-actin as the internal reference, qRT-PCR was carried out using SYBR^® Premix Ex TaqⅡ (Perfect Real Time; TaKaRa Code: DRR081, Japan) on ABI7500. DNA Amplification was performed under the following conditions: 95°C for 30s, followed by 40 cycles of 95°C for 5s, 60°C for 30s and 72°C for 30s. Melting curve analysis: 94°C for 60s; 37°C for 60s; 72°C for 120s. Experiments were performed in triplicates for the same reaction. The relative levels of lncRNA transcripts to the control β-actin were calculated by 2^{− ΔΔCt}.