Prism 7300 real time pcr system

Total RNA was extracted from lung, kidney and liver tissues and cultured macrophages with TRIzol® reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's protocol. cDNA was synthesized with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed by Prism 7300 Real Time PCR System (Applied Biosystems) using TaqMan Gene Expression Master Mix (Applied Biosystems) in a final reaction volume of 20 μl with each primer. The following primers were purchased from Applied Biosystems: mouse GAPDH (Mm 99999915_gl), mouse HMGB1 (Mm 00849805_gl), mouse TLR4 (Mm 00445274_m1), mouse TNF-α (Mm 00443258_ml) and mouse SOCS3 (Mm 00545913_s1). A negative control without cDNA did not produce any amplicons. Data were analyzed with 2^−ΔΔCt value calculation, using GAPDH for normalization.

Total cellular RNA was isolated from ~30 mg of pulverized gastrocnemius muscle using a FastRNA Green Kit (MP Bio, Santa Ana, CA, USA) and RNA-containing homogenates were further purified using PureLink RNA mini columns (Life Technologies, Carlsbad, CA, USA). Quantification of RNA was performed on each sample using spectrophotometry by measuring the absorbance at 260 nm using a Nanodrop 1000 (Thermo Scientific, Rockford, IL, USA). RNA purity was determined by a 260:280 nm ratio of ≥2.0 and the absence of organic contamination such as phenol and carbohydrates was verified by a 230:260 nm ratio of ≥1.8. RNA (1 μg), which was reverse transcribed (#4368814; Life Technologies, Carlsbad, CA, USA) and 25 ng of cDNA per reaction was used for real-time PCR with TaqMan gene expression assays (#4331182; Life Technologies, Carlsbad, CA, USA). Levels of MAFbx (Mm00499523_m1), MuRF-1 (Mm01185221_m1), and myostatin (Mm01254559_m1) mRNA were analyzed and normalized to TATA box binding protein endogenous control reference mRNA (Tbp; Mm01277042_m1) using a Prism 7300 real-time PCR system (Life Technologies, Carlsbad, CA, USA). Target mRNA levels were calculated using the ∆∆C_t method and expressed relative to control samples.

Library preparations were performed according to manufacturer's instructions, in 96-well MicroAmp Optical 96-Well Reaction Plates (Life Technologies) or Hard-Shell Low-Profile Thin-Wall 96-Well Skirted PCR plates (BioRad). Quality and band size of libraries were assessed using D1K and HS D1K Screen Tapes (Agilent) on a Tapestation 2200 (Agilent) at multiple steps during each protocol, typically after size selection and after PCR amplification. Libraries were quantified by qPCR using the Library Quantification Kit for Illumina sequencing platforms (KAPA Biosystems, Boston, USA), using a Prism 7300 Real Time PCR System (Life Technologies). Unless otherwise stated, libraries were normalised to a working concentration of 10 nM using the molarity calculated from qPCR adjusted for fragment size with the Tape Station median.