Lightcycler 480 instrument 2

For the ssa-miR-301a-3p, the first-strand cDNA was synthesized from the total RNA using Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Dalian, China). Specific primer for the ssa-miR-301a-3p was designed and primer sequences were listed in Table 1. The qRT-PCR was performed using Mir-X miRNA qRT-PCR SYBR Kit (TaKaRa, Dalian, China) and LightCycler® 480 Instrument II (Roche, Basel, Switzerland). U6 was used as an endogenous control. The cycling parameters were as follows: 95 °C for 10 s, 40 cycles at 95 °C for 5 s and 60 °C for 20 s; melting curve analyses at 95 °C for 60 s, 55 °C for 30 s, and 95 °C for 30 s.
For hsp90b2, the PrimerScript™RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) was used to synthesize first-strand cDNA, and SYBR® Premix Ex Taq kit (TaKaRa, Dalian, China) and LightCycler® 480 Instrument II were used for qRT-PCR. The ACTB was taken as a reference gene. Specific primer for the hsp90b2 was listed in Table 1. The cycling parameters were as follows: 95 °C for 10 s, 40 cycles at 95 °C for 5 s and 61 °C for 20 s; melting curve analyses at 95 °C for 60 s, 55 °C for 30 s, and 95 °C for 30 s.

After extracting the total RNA using TRIzol Reagent (Invitrogen), the total RNA was reverse transcribed into cDNA using Takara RT-PCR Master Mix (DRR0036A). The RT-qPCR system, LightCycler 480 Instrument II and SYBR Green solution (Takara) were used to detect the relative expression of the target gene, SMPDL3A using β-actin as an internal reference. The primers used in this study were synthesized by Sangon Biotech (Shanghai) Co., Ltd., China, with the following primer sequences: SMPDL3A forward primer: 5′-CTCACAGAGACAGCATTATGGTT-3′; SMPDL3A reverse primer: 5′-TTCACTGGTGTAACAGCAGGA-3′; β-actin forward primer: 5′-GGCGGCACCACCATGTACCCT-3′; β-actin reverse primer: 5′-AGGGGCCGGACTCGTCATACT-3′.