Quantstudiotm 3 real time pcr system

This variant was determined with a predesigned Taqman assay ID C__33641686_10 (Applied Biosystems), using a QuantStudio^TM 3 Real-Time PCR Systems following the manufacturer’s instructions. Homozygous G/G, heterozygous G/Del, and homozygous Del/Del genotype groups were defined.

Total RNA was extracted from cells using Trizol reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. cDNA was synthesized with 50 ng RNA using M-MuLV transcriptase (M0253S; New England BioLabs, Ipswich, MA, USA) according to the manufacturer’s protocols. qRT-PCR was performed using a Power SYBR^® Green PCR Master Mix (4367659; Applied Biosystems, Waltham, MA, USA) on QuantStudio^TM 3 Real-Time PCR systems (Applied Biosystems, Waltham, MA, USA). PCR conditions are as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The following primers were used for PCR reactions: PGC-1α (forward primer: 5′- GACACAACACGGACAGAA-3′, reverse primer: 5′-CACAGGTATAACGGTAGGTAA-3′), LARS1 (forward primer: 5′-GGACAGCCTTGCATGGATCAT-3′, reverse primer: 5′- TAGATGGGTATGGCTCAAGCA-3′), β-Actin (forward primer: 5′-CGATGCCCTGAGGCTCTTT-3′, reverse primer: 5′-AGTGATGCCACAGGATTCCA-3′). Each mRNA expression levels were normalized with β-Actin and the average relative changes were calculated using the $2^{-△△Ct}$ method [26 (link)]. All reactions were carried out in triplicate.

Total RNA was extracted from samples using the AllPrep DNA/RNA/Protein Mini Kit (#80004, Qiagen Sciences, Inc., Germantown, MD, United States) according to the manufacturer’s protocol. RNA was eluted with RNase-free water. Total RNA was quantified using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). For reverse transcription, 0.25 μg of total RNA was used in the SuperScript^TM IV First-Strand cDNA Synthesis System (#18091050, Thermo Fisher Scientific, Waltham, MA, United States) with a mixture of 50 μM oligo-dT and 50 ng/μl random hexamers as primers. Real-time primers were designed with Primer3web, version 4.4.0¹ and validated using melt curve analysis and agarose gel electrophoresis. PCR primers used in this study are shown in Supplementary Table 1. Significant gene expression was evaluated using the FastStart Universal SYBR Green Master (ROX; #4913850001, Roche Molecular Systems, Inc., Branchburg, NJ, United States) in triplicate in QuantStudio^TM 3 Real-Time PCR Systems (Thermo Fisher Scientific, Waltham, MA, United States). The relative quantity of mRNA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the comparative 2^–ΔΔCt method.