Extraction of RNA and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

Peripheral blood samples (5 ml) were collected in tubes containing ethylenediaminetetraacetic acid (EDTA) from each subject. PBMCs were purified from peripheral blood by Ficoll-Hypaque density gradient centrifugation. Total RNA was extracted from PBMCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and the concentrations of RNA were measured by a NanoDrop™ 2000 spectrophotometer (Thermo Scientific, USA).

Total RNA were reverse-transcribed into cDNA by the PrimeScript^TM RT reagent Kit (Takara Bio Inc, Japan). To determine the expression level, quantitative real-time PCR (qPCR) with SYBR Green (SYBR® Premix Ex Taq™ II,Takara Bio Inc, Japan) was performed using an ABI ViiA™ 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Cycle conditions were as follows: 95 °C for 1 min, followed by 42 cycles at 95 °C for 10 sec, 60 °C for 30 sec and 72 °C for 1 min. The lncRNA expression was determined by comparison with housekeeping gene β-actin from the same sample as internal control. The primer sequences used for qPCR are given in Table ^S9. The relative expression of lncRNAs were calculated using 2^−△△Ct method normalized to endogenous control³⁷.