Stepone plus real time polymerase chain reaction system

Frozen gastrocnemius muscle was homogenized using Sepasol-RNA I Super G (Nacalai Tesque, Inc., Kyoto, Japan), total RNA was extracted using an RNeasy Fibrous Mini kit (Qiagen, Valencia, CA, USA), and TaqMan real-time polymerase chain reaction with a StepOnePlus real-time polymerase chain reaction system (Thermo Fisher Scientific) was used to measure the mRNA levels of MuRF1(Mm01185221_m1), Atrogin1 (Mm00499523_m1), Bax (Mm00432051_m1), Bcl-2 (Mm00477631_m1), C1qa (Mm07295529_m1), Axin2 (Mm00443610_m1), Agtr1a (Mm01957722_s1), angiotensinogen (Mm0599662_m1) and 18S rRNA (Mm03928990_g1).

Polymorphisms in the RNF213 gene was determined as previously reported [32 (link)]. Single nucleotide polymorphism (SNP) for rs112735431 (https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=112735431) was analyzed using the TaqMan SNP genotyping assay (Assay ID: C_153120198_10; Applied Biosystems, Foster City, CA, USA) on a StepOnePlus real-time polymerase chain reaction (PCR) system (Applied Biosystems, Foster City, CA, USA). The basic cycling parameters were as follows: hold at 95°C for 10 minutes, followed by 40 cycles of PCR amplification comprising of denaturation at 95°C for 15 seconds, and annealing and extension at 60°C for 1 minute. Genotype calls were evaluated with Applied Biosystems TaqMan Genotype software. The investigators who assessed genotype were blinded to phenotypic information.

Samples were diluted to 5ng/μl and transcribed into cDNA using a Taqman Advanced MiRNA cDNA Synthesis Kit from Applied BioSciences. The cDNA underwent an additional amplification step to increase yields of unstable miRNAs (MiR-Amp). Samples were diluted 1:10 and loaded onto qPCR plates with Taqman Fast Advanced Master Mix. Each sample received 2 qPCR runs using the StepOnePlus Real Time polymerase chain reaction (PCR) System (Applied Biosystems, Foster City, CA), including one to evaluate for U6 a small non-coding spliceosome RNA that is a common endogenous control (Campos-Melo et al., 2013 (link)). The next qPCR run was with the custom miRNA plates with primers for selected targets. Samples were tested in duplicate.

The dominant hydrogenase gene hup, formate dehydrogenase gene fdh, and RDase gene tceA were quantified. For hup and fdh, amplification was performed using a Step One Plus Real-Time Polymerase Chain Reaction (PCR) System (Applied Biosystems, United States) with SYBR green-based detection agents (Morris et al., 2006 (link)). The reaction solution contained 7.5 μL of SYBR Green mix (DBI Bestar, Ludwigshafen, Germany), 0.5 μL of Rox, 0.5 μL of each primer, 2 μL of DNA template, and 4 μL of double-distilled water. The program was run for 10 min at 95°C for Taq activation, followed by 40 cycles of 15 s at 95°C, 60 s at 55°C and a melting curve stage from 55 to 95°C. For the tceA gene, the method was the same as that described by Ritalahti et al. (2006) (link). The method limit of quantitation was 10³ copies/mL. The sequences of primers and probes were synthesized by Sangon Biotech, as shown in Supplementary Table 1. The standard curves and amplification efficiency are shown in Supplementary Table 2.