Abstract
The use of banked human tissue, obtained with informed consent after elective surgical procedures, represents a powerful model for understanding underlying mechanisms of diseases or therapeutic interventions and for establishing prognostic markers. However, donated tissues typically have varying times of warm ischaemia in situ due to blood arrest or cold ischaemia due to procurement and transportation. Hence, before using these tissues, it is important to carry out pre-analytical studies to ensure that they are representative of the in vivo state. In particular, tissues of the gastrointestinal tract have been thought to have low RNA stability. Therefore, this study aimed to determine if extended warm or cold ischaemia times and snap-freezing or banking in RNA stabilization solution affects RNA integrity or gene expression in human ileum mucosa. In short, ileum mucosa was collected for up to 1.5 h and 6 h of simulated warm or cold ischaemia respectively. Subsequently, RNA integrity and gene expressions were determined. It was found that RNA integrity remained high over the course of warm and cold ischaemia examined and there were in general no significant differences between snap-freezing and banking in RNA stabilization solution. Following the same trend, there were in general no significant changes in gene expressions measured (MYC, HIF1α, CDX, HMOX1 and IL1β). In conclusion, RNA in the ileum mucosa is maintained at a high integrity and has stable gene expression over the examined time course of warm or cold ischaemia when banked in RNA stabilization solution or snap-frozen in liquid nitrogen. As the average warm and cold ischaemia times imposed by surgery and the process of tissue banking are shorter than the time period examined in this study, human ileum mucosa samples collected after surgeries could be used for gene expression studies.
Highlights
Comparative quantification of gene expression, through real-time quantitative polymerase chain reaction (RT-qPCR) or gene expression microarrays, is essential for understanding the molecular bases of diseases
When RNA Integrity Number (RIN) was plotted against warm ischaemia time (Fig 5C) or cold ischaemia time (Fig 5D), there were generally no significant differences between
Due to the nature of the surgery described in the method section above, ileum samples could be obtained in the operation room without warm ischaemia in situ
Summary
Comparative quantification of gene expression, through real-time quantitative polymerase chain reaction (RT-qPCR) or gene expression microarrays, is essential for understanding the molecular bases of diseases. For such studies, human tissue banks such as the one at the Surgical Clinic in the Hospital of the University of Munich, provide powerful models by banking leftover tissue in excess of what is required for diagnostic tests by the pathologists. To our knowledge, the effects of the above conditions on ileum mucosa have not been examined This is of interest as previous studies have shown that RNA was least stable in tissues of the gastrointestinal tract [2, 5] possibly due to the presence of ribonucleases (RNases), which play a role in epithelial host defence [6,7,8,9]
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