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One‐Step Real Time RT‐PCR for the Detection of Exercise‐Inducing Circulating MicroRNAs in Sedentary Males
Author(s) -
Chen KuanMing Jacky,
Huang ChingHui,
Lin KeTing,
Wang JongShyan
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.839.19
Subject(s) - microrna , skeletal muscle , real time polymerase chain reaction , medicine , biology , gene , biochemistry
Circulating microRNAs (c‐miRNAs) derived from endothelium and skeletal muscle can response to acute exercise and exercise training. However, the commonly used protocols are usually costly and time consuming. Hence, this study developed a four‐primers‐based miRNA quantification method, and simultaneously performed both reverse transcription and PCR by a one‐step operation on a real‐time system. Moreover, we designed the specific primers for two “exercise‐inducing” c‐miRNAs: Hsa‐miR‐133a‐3p is distinctly expressed in cardiac and skeletal muscles and hsa‐miR‐126‐3p is dominant expressed in endothelium. Additionally, we also developed primers targeting to the synthetic internal control miRNA, cel‐miR‐39‐3p, which is added during RNA extraction. The results showed that excellent linearity (R 2 =0.9998) between the Ct values and the logarithms (log) of miRNA amount was observed in the range from 6.1 attomole to 6.3 femtomole of cel‐miR‐39‐3p. Furthermore, maximal graded exercise significantly increased has‐mir‐126‐3p (52±28%, p<0.05) and hsa‐miR‐133a‐3p (113±35%, p<0.05) in healthy sedentary males (n=12). Therefore, we conclude that one step RT‐qPCR is reliable and efficient, and has the superiority in the high throughput routing assay of c‐miRNAs. According to the methodology, acute strenuous exercise increases the releases of muscle/endothelium‐related miRNAs into the peripheral blood compartment in sedentary males.