Sensiscript reverse transcriptase

The level of cytokines in lung homogenates and culture supernatants was determined by Kit enzyme-linked immunosorbent assay (R&D Systems, Milan, Italy). Real-time Reverse Transcriptase-PCR was performed using the iCycleriQdetection system (Bio-Rad, Segrate (MI), Italy) and SYBR Green chemistry (Finnzymes Oy, Espoo, Finland). Cells were lysed and total RNA was extracted using RNeasy Mini Kit (QIAGEN, Milan, Italy) and was reverse transcribed with Sensiscript Reverse Transcriptase (QIAGEN) according to the manufacturer's directions. PCR primers for transcription factors were used as described.^{30 (link)} The following c-maf primers were used: sense 5′-GTAGACCACCTCAAGCAGGA-3′ antisense 3′-GAAAAATTCGGGAGAGGAAG-5′. Amplification efficiencies were validated and normalized against Gapdh. The thermal profile for SYBR Green real-time PCR was at 95 °C for 3 min, followed by 40 cycles of denaturation for 30 s at 95 °C and an annealing/extension step of 30 s at 60 °C. Each data point was examined for integrity by analysis of the amplification plot. The mRNA-normalized data were expressed as relative cytokine mRNA expression in treated cells compared with that of unstimulated cells.

Total RNA was isolated from diaphragm and quadriceps muscle tissue using RNeasy (Qiagen, Hilden, Germany) and reverse transcribed into complementary DNA using random hexamers and Sensiscript reverse transcriptase (Qiagen, Hilden, Germany). An aliquot of the complementary DNA was used for quantitative reverse transcription PCR applying the LightCycler™ (Roche Diagnostics Inc). The expression of specific genes was normalized to the expression of 18S ribosomal RNA. The following primers and conditions were used 18S ribosomal RNA: 5′-ATACAGGACTCTTTCGAGGCCC-3′ and 5′-CGGGACACTCAGCTAAGAGCAT-3′ at 62°C annealing; IGF-1: 5′-TCTACCTGGCACTCTGCTTGCT-3′ and 5′-CTGAGTCTTGGGCATGTCAGTG-3′ at 62°C annealing.

Total RNA was isolated from C2C12 cells and reverse transcribed into cDNA using random hexamers and Sensiscript Reverse Transcriptase (Qiagen, Hilden, Germany). An aliquot of the cDNA was used for quantitative RT‐PCR, applying the Light Cycler system (Roche Diagnostics, Mannheim, Germany). The expression of specific genes was normalized to the expression of hypoxanthin phosphoribosyltransferase mRNA. For quantification of MuRF1 expression, fluorescence resonance energy transfer technology was applied using the following primers (TIB MolBiol, Berlin, Germany) and conditions: hypoxanthin phosphoribosyltransferase: 5′‐CTCATggACTgATTATggACAggAC‐3′ and 5′‐gCAggTCAgCAAAgAACTTATAgCC‐3′, 60°C annealing; MuRF1: 5′‐gATgTgCAAggAACACgAA‐3′, 5′‐CCTTCACCTggTggCTATTC‐3′, LC640‐gCACAAggAgCAAgTAggCACCTCAC‐PH, 5′‐gCCTggTgAgCCCCAAACACCT‐FL, annealing 58°C.