Takara primescript reagent kit

cDNA was synthesized from 1 μg total RNA using a Takara PrimeScript Reagent kit (TakaraBio, Kusatsu, Shiga, Japan) per the manufacturer’s instruction. Real-time PCR was conducted using a BioRad PCR Thermal Cycler with a final reaction volume of 25 μL containing 50 ng cDNA, 1 μL of each primer pair (20 μM), and Takara TB Green Premix Ex Taq II (TII RNaseH Plus), as per the manufacturer’s kit protocol to determine the relative mRNA concentration for each gene of interest under each treatment condition; all reactions were performed in duplicate. The sequences of the primers used for the PCR reactions are shown in Supplementary Table S2. The relative RNA concentrations were determined using a standard curve generated using a preamplified PLIN4 PCR product or GAPDH. The PCR reaction conditions were as follows: 95 °C for 10 s, followed by 40 cycles of 95 °C for 20 s, 64 °C for 20 s, and 72 °C for 10 s.

Total RNA was isolated from the peripheral blood leukocytes of the proband and control individuals by TRIzol LS reagent (Invitrogen, CA, United States). RNA was reverse-transcribed using a TaKaRa PrimeScript reagent kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol. To investigate the abnormal splicing of the variant of PATL2, we amplified PATL2 cDNA using primers spanning exons 8 to 15 in the proband and a normal control individual (NC). Then, the obtained PCR products were analysed by gel electrophoresis on a 1% agarose gel, and the further Sanger sequencing was performed on the cDNA amplified product. The PCR products were sequenced using ABI BigDye3.1 (Applied Biosystems, Foster City, CA, USA) and analysed using an ABI 3730XL sequencer.