Reverse transcription and quantitative real-time-PCR
APPENDIX
For reverse transcription, the miRCURY LNA RT Kit (Qiagen, Hilden, Germany) was used and quantitative real-time PCR was performed using miRCURY LNA miRNA PCR assays (Appendix A) in a 10 µl reaction containing 3 µL of cDNA (1:30) and a CFX real-time PCR detection system (BioRad, Munich, Germany). Assays were performed in duplicate. The amount of the respective miRNA was normalized to miR-425-3p (previously determined as being stably expressed pre and post training intervention) and cel-miR-39 (spike-in control). A Ct cut-off of 35 was set as the lower limit of detection and the fold change was calculated as (2 − ΔΔ