正确的生物标记对于获得可靠的数据至关重要,特别是对于下一代测序方法。病变血管组织,具有扩展的动脉粥样硬化病理,由于RNA质量低,代表了一个特殊的挑战。为了解决这个问题,我们从瑞士血管生物库(SVB)收集的血管样本中分离出RNA;这些样本包括腹主动脉瘤(AAA),外周动脉疾病(PAD),健康的主动脉(HA),和肌肉样本。我们用了不同的方法,调查了各种入院解决方案,确定的RNA完整性数(RIN),并进行了管家基因的表达分析(ACTB,GAPDH),核糖体基因(18S,28S),和长链非编码RNA(MALAT1,H19)。我们的结果表明,来自患病血管组织的RIN较低(2-4)。如果打算分离原代细胞,就像我们的SVB一样,冷冻保护溶液是比RNAlater更好的组织保存选择。因为RNA降解是随机进行的,建议使用具有相似RIN的控件。否则,数据可能传达RNA降解的差异,而不是相应基因的表达。此外,由于患病血管样本中的18S和28S基因被降解并与低RIN相对应,我们认为代表总RNA的崩解状态的DV200,是选择用于组学分析的样本的更好的决策帮助。
Proper biobanking is essential for obtaining reliable data, particularly for next-generation sequencing approaches. Diseased vascular tissues, having extended atherosclerotic pathologies, represent a particular challenge due to low RNA quality. In order to address this issue, we isolated RNA from vascular samples collected in our Swiss Vascular Biobank (SVB); these included abdominal aortic aneurysm (AAA), peripheral arterial disease (PAD), healthy aorta (HA), and muscle samples. We used different methods, investigated various admission solutions, determined RNA integrity numbers (RINs), and performed expression analyses of housekeeping genes (ACTB, GAPDH), ribosomal genes (18S, 28S), and long non-coding RNAs (MALAT1, H19). Our results show that RINs from diseased vascular tissue are low (2-4). If the isolation of primary cells is intended, as in our SVB, a cryoprotective solution is a better option for tissue preservation than RNAlater. Because RNA degradation proceeds randomly, controls with similar RINs are recommended. Otherwise, the data might convey differences in RNA degradation rather than the expressions of the corresponding genes. Moreover, since the 18S and 28S genes in the diseased vascular samples were degraded and corresponded with the low RINs, we believe that DV200, which represents the total RNA\'s disintegration state, is a better decision-making aid in choosing samples for omics analyses.