Journal of IiMER Volume 10 Issue 1 a comparative cohort of multiple sclerosis (MS) patients, as an example of a ‘fatigue illness’ distinct from ME/CFS. Our goal is to examine and correlate changes in plasma cytokines and miRNAs, with the cellular transcriptome, and the proteome in purified immune cells. In the second study we are planning to link these molecular analyses to an exercise regime that measures physiological parameters before and after exercise. These two pilot studies in turn are preliminary to a planned larger study with 40 carefully evaluated patients from Dr Vallings health practice. To understand the disease mechanism of ME/CFS in our NZ cohort at a molecular level, June 2016 coding, small and large noncoding RNAs, and initiating a study of the proteome. We have become acutely aware that the key to validating results from small patient studies, whether it be for rare diseases or the pilot studies we are conducting, are appropriate biostatistical analyses. This is a dynamic field and new ways of analysing data are constantly being developed that can extract richer information from low numbers of patients, providing random variables outside the study design can be controlled. It is sobering a comprehensive analysis has been undertaken in an initial pilot study group. Peripheral blood samples were taken from the study participants and plasma, lymphocytes and neutrophils extracted. From the plasma and lymphocyte immune cells total RNA (including small RNAs) was isolated using a mirVana™ PARIS™ RNA and Native Protein Purification Kit. Total protein was also obtained from the lymphocyte and neutrophil cells for western and proteome analysis. So far we have obtained and analysed data on both cytokines and microRNAs from plasma in one of our pilot studies, and are awaiting the results of analyses on the transcriptome encompassing that of the 750 000 studies that have suggested statistically significant leads for therapy development only a handful have stood up to rigorous testing and development17. That is a challenge for us in taking a ‘precision medicine’ approach to these studies. In consultation with an experienced biostatistician we are exploring a number of mainstream statistical approaches in an attempt to ensure apparent molecular differences between ME/CFS patients and controls have validity. There is an advantage in having two independent statistical tests so that if an effect is found with one test it can be confirmed with the other. Since our studies are discovery tests, Invest in ME (Charity Nr. 1114035) www.investinme.org Page 50 of 77

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