Evaluation of sampling methodologies for use in investigation of the health benefits of community-based exercise
Thwin, Phway Phway
Cardiff Metropolitan University
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Low- intensity exercise exerts therapeutic benefits on molecular pathways controlling reverse cholesterol transport, arterial stiffness and monocyte polarisation via transcription factors peroxisome proliferator-activated receptor-y (PPARy) activation. This study developed and optimised assay systems for measuring exercise- induced changes in the PPARy signalling mechanisms in long-term PhD project "translating exercise-derived health benefits from laboratory to community" and in future exercise-based studies. Firstly, scavenger receptor CD36, PPARy and adenosine tri-phosphate (ATP)-binding cassette transporter A1 (ABCA1) genes expression in monocytes and buffy coat samples were measured using real-time polymerase chain reaction (RT-PCR) to investigate the types of sample in which genes-of-interest were optimally expressed. Secondly, ribonucleic acid (RNA) isolation and complementary deoxyribonucleic acid (cDNA) synthesis were performed using Ambion® TRI reagent®, µMACS™ mRNA isolation and µMACS™ One-step cDNA kits to find out the best protocol that produced maximal RNA yields of PPARy- regulated genes. Thirdly, current study analysed four variable sample sizes of mononuclear cells using RT-PCR and flow cytometry to determine minimum cell volume whereby effects of exercise-mimic on expression of PPARy- targeted genes and proteins were optimally observed. Finally, assays performance of RT-PCR and flow cytometry with regard to measuring effects of exercise mimic on PPARy-regulated signalling axis was evaluated. Monocytes sample produced significantly increased levels of CD36, PPARy and ABCA1 (CD36, ~ 2.1- fold; PPARy, ~5.6- fold; ABCA1, ~2.3- fold) compared to buffy layer sample. Among RNA isolation and cDNA synthesis methods investigated, CD36 cDNA levels generated by both µMACS™ One-step cDNA kit and Ambion® TRI reagent® were significant higher than those obtained by µMACS™ mRNA isolation column. This study detected sample size of 0.8 x 106 cells/ sample as minimum requirement to observe reliable and marked up-regulation of CD36 in mononuclear cells after treatment with exercise mimic (the PPARy activator, rosiglitazone). Both RT-PCR and flow cytometry showed equal sensitivity and were able to detect exercise mimic-induced dramatic CD36 upregulation accurately. In conclusion, this study provided efficient assay systems to be applied in subsequent work (e.g. UWIC-based PhD project for assessing the effects of exercise on PPARy-mediated signalling pathways), and in other prospective exercise-based studies.
MSc Biomedical Sciences
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