Journal of IiMER Volume 10 Issue 1 and were used to convert the miRNA to cDNA. To improve assay sensitivity and account for the limited abundance of some miRNAs, a preamplification step using Megaplex™ PreAmp Primers was included. Data was assessed using the BioC/R package HTqPCR (high-throughput qPCR)25. MiRNAs with ‘undetermined’ ct values or ct values >35 were removed from the analysis. A norm rank invariant method was used to normalise the data. The two study groups have initially been compared using a nonparametric Mann Whitney U test to determine statistically significant miRNAs. To complete our molecular study of the pilot study group we are also completing full transcriptome and proteome analyses of the ME/CFS and healthy control individuals. At present, as stated above, transcriptome analysis of the 20 participants in our first pilot study is underway. Total RNA was extracted from lymphocytes purified from the original blood samples, using the same extraction kit as for the miRNA analysis. We are investigating by HiSeq analysis both small RNA (NEXTflex small RNA libraries), including miRNA, and total RNA (TruSeq stranded total RNA libraries (ribozero human/rat/mouse). The proteome analysis will analyse total protein extracts from the patient and control subject’s lymphocyte cells. Samples will be compared by sensitive global proteomics profiling using the mass spectrometry facilities in the Centre for Protein Research, University of Otago. The approach will allow for a semi-quantitative comparison of protein profiles using for example spectral counts or extracted peak intensities. It will be of significant interest to see if the results of these June 2016 analyses correlate in any way with our miRNA and cytokine investigations to suggest disease mechanisms or pathways. Invest in ME (Charity Nr. 1114035) www.investinme.org Our second pilot study conducted by exercise physiologist, Dr Lynette Hodges at Massey University, aims to link exercise performance with molecular analyses. The exercise part of the study has examined differences in fatigue parameters among three study groups, ME/CFS patients, MS patients and healthy controls. Each group performs repeated incremental exercise tests separated by 24 hours. Incremental exercise tests utilised an increased power requirement each minute and measured oxygen consumption, carbon dioxide production, respiratory exchange ratio, and power output, among other parameters. Each incremental exercise test was followed by a steady state 5 min exercise test conducted at the anaerobic threshold, measuring cardiac output. In contrast to the other two groups ME/CFS patients uniquely had lower power output on the day 2 reexercise test, consistent with the reported characteristic of the disease, post-exertional malaise. Cytokine analysis data on plasma samples from these subjects have just been collected. Applying miRNA analysis in this collaborative exercise study is appealing; it has been shown that circulating miRNA-signatures are distinct enough to enable discrimination between eccentric and concentric exercise23 and so they also may provide valuable indicators of the post-exertional malaise experienced by ME/CFS sufferers. It is hoped that we can also add data on the expression of coding genes, and of small and larger noncoding RNAs, together with altered protein profiles to give a more Page 52 of 77
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