He Yinli, Dong Lele, Yi Hongyang, Zhang Linpei, Shi Xue, Su Lin, Gan Baoyu, Guo Ruirui, Wang Yawen, Luo Qinying, Li Xiaojiao
BioBank, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China.
Department of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China.
Front Genet. 2023 Jan 4;13:1091685. doi: 10.3389/fgene.2022.1091685. eCollection 2022.
Genetic and epigenetic reprogramming caused by disease states in other tissues is always systemically reflected in peripheral blood leukocytes (PBLs). Accurate transcriptional readouts of Messenger RNA (mRNA) and Long non-coding RNA (lncRNA) in peripheral blood leukocytes are fundamental for disease-related study, diagnosis and treatment. However, little is known about the impact of preanalytical variables on RNA quality and downstream messenger RNA and Long non-coding RNA readouts. In this study, we explored the impact of RNA extraction kits and timing of blood placement on peripheral blood leukocyte-derived RNA quality. A novel enhanced evaluation system including RNA yields, purity, RNA integrity number (RIN) values and β-actin copies was employed to more sensitively identify RNA quality differences. The expression levels of informative mRNAs and Long non-coding RNAs in patients with chronic obstructive pulmonary disease (COPD) or triple-negative breast cancer (TNBC) were measured by Quantitative reverse transcription polymerase chain reaction (qRT-PCR) to investigate the impact of RNA quality on transcriptional readouts. Our results showed that the quality of RNA extracted by different kits varies greatly, and commercial kits should be evaluated and managed before batch RNA extraction. In addition, the quality of extracted RNA was highly correlated with the timing of blood placement, and the copy number of β-actin was significantly decreased after leaving blood at RT over 12 h. More importantly, compromised RNA leads to skewed transcriptional readouts of informative mRNAs and Long non-coding RNAs in patients with chronic obstructive pulmonary disease or triple-negative breast cancer. These findings have significant implications for peripheral blood leukocyte-derived RNA quality management and suggest that quality control is necessary prior to the analysis of patient messenger RNA and Long non-coding RNA expression.
其他组织中的疾病状态所引起的基因和表观遗传重编程总会在外周血白细胞（PBLs）中得到全身性反映。外周血白细胞中信使核糖核酸（mRNA）和长链非编码核糖核酸（lncRNA）的准确转录读数对于疾病相关研究、诊断和治疗至关重要。然而，关于分析前变量对RNA质量以及下游信使核糖核酸和长链非编码核糖核酸读数的影响，人们了解甚少。在本研究中，我们探究了RNA提取试剂盒以及血液放置时间对外周血白细胞来源的RNA质量的影响。我们采用了一种新型的增强评估系统，该系统包括RNA产量、纯度、RNA完整性数值（RIN）以及β-肌动蛋白拷贝数，以便更灵敏地识别RNA质量差异。通过定量逆转录聚合酶链反应（qRT-PCR）测定慢性阻塞性肺疾病（COPD）或三阴性乳腺癌（TNBC）患者中信息性mRNA和长链非编码RNA的表达水平，以研究RNA质量对转录读数的影响。我们的结果表明，不同试剂盒提取的RNA质量差异很大，在批量提取RNA之前应对商业试剂盒进行评估和管理。此外，提取的RNA质量与血液放置时间高度相关，室温放置超过12小时后β-肌动蛋白的拷贝数显著降低。更重要的是，受损的RNA会导致慢性阻塞性肺疾病或三阴性乳腺癌患者中信息性mRNA和长链非编码RNA的转录读数出现偏差。这些发现对外周血白细胞来源的RNA质量管理具有重要意义，并表明在分析患者信使核糖核酸和长链非编码核糖核酸表达之前进行质量控制是必要的。
