RNA preparation and RT-qPCR

RNAzol reagent (Sigma–Aldrich, St. Louis, MO, U.S.A.) was mixed with plasma to extract total RNAs. Total RNAs were reverse-transcribed to synthesize cDNA using SSRT II system (Thermo Fisher Scientific) by incubating at 25°C for 5 min, 55°C for 10 min, and 75°C for 5 min. To detect the expression of SNHG1, Applied Biosystems™ PowerUp™ SYBR™ Green Master Mix was used to prepare PCR systems, and PCRs were perfromed with 18S RNA as endogenous control. Primer sequences were: 5′-AGGCTGAAGTTACAGGT-3′ (forward) and 5′-TTGGCTCCCAGTGTCTT-3′ (reverse) for SNHG1; 5′-TACCACATCCAAGGAAGCA-3′ (forward) and 5′-TTTTCGTCACTACCTCCCCG-3′ (reverse) for 18S rRNA. Expression of SNHG1 of normalized to 18S RNA according to 2^−ΔΔC_T method. PCR products were sequenced to make use corrected products were obtained. There replicates were performed for each reaction.