Abstract
This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue.
Highlights
Despite improvements in coronary revascularisation techniques, ischaemic heart disease (IHD) remains a leading cause of morbidity and mortality in more economically developed countries,[1] and patients are at risk of developing post-intervention complications.[2]
Our results indicate that for technically demanding and costly applications such as RNA sequencing or other high-throughput screening methods, it is critical to validate the RNA extraction method for each sample type, as no single method is optimal for all
Ip et al compared an organic extraction method with a solid-phase extraction method on archived right atrial appendage samples,[50] these samples are different in size and composition from the more clinically-relevant left ventricular biopsies obtained in the ARCADIA study
Summary
Despite improvements in coronary revascularisation techniques, ischaemic heart disease (IHD) remains a leading cause of morbidity and mortality in more economically developed countries,[1] and patients are at risk of developing post-intervention complications.[2]. NIHR Biomedical Research Centre at University Hospitals Bristol NHS Foundation Trust and the University of Bristol. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research, or the Department of Health and Social Care. The ARCADIA study was designed and delivered in collaboration with the Clinical Trials and Evaluation Unit (CTEU), a UKCRC registered clinical trials unit which, as part of the Bristol Trials Centre is in receipt of National Institute for Health Research CTU support funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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