Thermal cycler system

After obtaining informed consent, we collected 5 mL EDTA-anticoagulated blood sample from study subject. PBMCs were freshly isolated from peripheral blood within 4 h and stored at −80 °C. Total RNAs were extracted from PBMCs using TRIzol reagent. In addition, the concentrations and purity of these total RNA samples were measured by NanoDrop™2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). All of the qualified total RNAs were reverse-transcribed into cDNA based on instructions of the Prime Script TM RT reagent Kit (Takara Bio Inc, Shiga Prefecture, Japan), and stored at −80 °C for further detection.
The qRT-PCR was carried out in duplicate in an optical 96-well plate with a MyCyclerTM Thermal Cycler system by using ABI ViiA™ 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The qRT-PCR primer sequences are listed in Table 1. Housekeeping gene β-actin was used as internal control. According to specifications of SYBR Green (SYBR^® Premix Ex Taq™ II, Takara Bio Inc, Shiga Prefecture, Japan), 0.4 μL forward primer and 0.4 μL reverse primer (10 μM) were applied into qRT-PCR reaction system. Thermal cycling conditions were as follows: 95 °C for 1 min, followed by 42 cycles at 95 °C for 10 s, 60 °C for 30 s and 72 °C for 1 min. 2−∆∆Ct method normalized to endogenous control was used for calculating the relative expression levels of lncRNAs.

Genomic DNA was extracted from peripheral blood using standard salting-out method. The primer sequences, annealing temperatures, and restriction enzyme used for each reaction are shown in Table 2. PCR was carried out in a thermal cycler system (Applied Biosystems, CA, USA) using a commercial Master mix (Kawsar Biotech Company, Iran). The PCR program included a primary denaturation at 95ºC for 5 minutes, followed by 35 cycles of 95ºC for 45 seconds, specific annealing temperatures for 35 seconds, and 72ºC for 60 seconds, with the final extension of 72ºC for 5 minutes. All LDLR PCR products were sequenced using an ABI 3100 DNA Sequencer (Applied Biosystems, CA, USA). For R3500W analysis, PCR products were then incubated with the restriction enzyme TaaI (HPYCH4III) (Fermentas, Lithuania) at 65 °C for 16 h. If C nucleotide exists at the polymorphic site, this enzyme will cut 267-bp PCR products into two 163 and 104 fragments. If T nucleotide exists, PCR product remains intact.