QFM measurements of TGF-β1 were validated by comparing results with ELISA and qPCR measurements of muscle homogenates from PAD-II and CTRL patients (N = 13 in each group). TGF-β1 protein expression was measured as part of a customized Human Inflammatory Cytokines Multi-Analyte ELISArray Kit (Qiagen, Valencia, CA, USA).
To measure TGF-β1 gene transcripts in skeletal muscle biopsies, RNA extraction, reverse transcription reactions, and qPCR were performed as previously described [40 (link), 42 (link)] and levels were normalized to myosin gene transcripts. Intrasession reliability was determined by comparing the TGF-β1 measurement of each slide from the mean of its duplicate pair. Intersession reliability was determined from the averages of patient biopsy specimens, analyzed a second time in the next analytical session. Detailed methodologies and validation results can be found in “Additional file1 ”.
To measure TGF-β1 gene transcripts in skeletal muscle biopsies, RNA extraction, reverse transcription reactions, and qPCR were performed as previously described [40 (link), 42 (link)] and levels were normalized to myosin gene transcripts. Intrasession reliability was determined by comparing the TGF-β1 measurement of each slide from the mean of its duplicate pair. Intersession reliability was determined from the averages of patient biopsy specimens, analyzed a second time in the next analytical session. Detailed methodologies and validation results can be found in “Additional file
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