Validation of Reference Genes for the Relative Quantification of Gene Expression in Human Epicardial Adipose Tissue

BACKGROUND: Relative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study was to evaluate and identify optimal reference genes for use in the relative quantification of gene expression in human epicardial fat depots of lean, overweight and obese subjects. METHODOLOGY/PRINCIPAL FINDINGS: Some of the commonly used reference genes including 18S, ACTB, RPL27, HPRT, CYCA, GAPDH, RPLPO, POLR2A and B2M were quantified using real-time PCR analysis. The expression stability of these genes was evaluated using Genorm, Normfinder and Bestkeeper algorithms. In addition, the effect of sample size on the validation process was studied by randomly categorizing subjects in two cohorts of n = 2 and n = 33. CONCLUSIONS/SIGNIFICANCE: CYCA, GAPDH and RPL27 were identified as the most stable genes common to all three algorithms and both sample sizes. Their use as reference gene pairs might contribute to the enhanced robustness of relative quantification in the studies involving the human epicardial adipose tissue

M146 Validation Of Five Non-invasive Respiratory Rate Monitors In Patients With Copd In A Laboratory Setting

Introduction There is a need of innovative models of care for patients with severe COPD and frequent AECOPD, and Telehealth (TH) is part of these programs. But current systems are limited by the parameters feasibly monitored in a domestic setting and lack of a reliable method of predicting exacerbations. Evidence from hospital based studies show that breathlessness increases during exacerbations. If respiratory rate (RR) could be reliably monitored remotely it may provide a significant advance in predicting and identifying COPD exacerbations and monitoring recovery. The aim of this study is to validate five non-invasive RR monitors (M1 to M5) in patients with COPD in a laboratory setting against a gold standard measurement of RR. Methods and results Five RR monitors identified in the literature were selected for validation against RR measured with a gold standard method (Oxycon mobile, Carefusion) in 23 patients with COPD (13 males, age 70 ± 8.3 years, FEV1 58.3 ± 17.1%pred) during a 52 min protocol of a total of 19 activities of daily living (i.e sitting, standing, walking at different speeds, climbing stairs, lifting objects and sweeping the floor). Patients wore simultaneously the five monitors and the Oxycon mobile and RR was recorded breath by breath and averaged by minute. One minute of each activity was selected for analysis using Bland and Altman plots. Bias and limit of agreement (LoA) was established for each monitor (Figure 1). Bias and LoA for the five monitors were the following (M1 2.15 (-17.9 to 22.2), M2 3.1 (-8.7 to 14.9), M3 2.2 (-12.12 to 16.6), M4 -2.5 (-11.7 to 6.8) and M5 -1.9 (-10.8 to 6.9)). Patients were compliant with the use of the five monitors. Conclusions Monitoring RR is feasible and non-intrusive in patients with COPD. We have identified two monitors (M4 and M5) with the lowest bias and the narrower LoA. These monitors will be further investigated in a home setting