QIAGEN GmbH, Hilden, Germany.
Clin Chim Acta. 2012 Apr 11;413(7-8):779-86. doi: 10.1016/j.cca.2012.01.015. Epub 2012 Jan 21.
Although important improvements of downstream molecular in vitro diagnostics assays based on RNA from blood were made, the pre-analytical workflow is still poorly defined.
We performed a multicenter study within the EU-granted SPIDIA project to investigate blood collection and shipping influence on the following RNA quality parameters: yield, purity, integrity, RT-qPCR interference and IL1B, IL8, FOS and GAPDH gene expression. Two models were designed: Exp A. Ten laboratories collected blood from an own donor into two different tubes (with or without stabilizer) and extracted RNA at two different times; Exp B. Blood was drawn from a single donor and shipped to ten laboratories in two different tubes (with or without stabilizer) for RNA extraction.
In both models and collection tubes, reliable results were obtained for purity, yield, GAPDH expression, and interferences. A substantial variation in RIN (Exp A) and in transcription levels of IL1B, IL8 and FOS (Exp B) was observed for blood collected in tube without stabilizer tubes. Overall the variability was higher among data obtained from unstabilized blood samples.
We defined the experimental setup for a larger ring trial throughout Europe. The chosen downstream analyses verified their potential, serving as adequate markers to test the quality of blood RNA.
尽管基于血液 RNA 的下游分子体外诊断检测有了重要的改进，但预分析工作流程仍未得到很好的定义。
我们在欧盟资助的 SPIDIA 项目中进行了一项多中心研究，以调查血液采集和运输对以下 RNA 质量参数的影响：产量、纯度、完整性、RT-qPCR 干扰以及 IL1B、IL8、FOS 和 GAPDH 基因表达。设计了两种模型：实验 A. 十个实验室从同一个供体收集血液到两个不同的管中（有或没有稳定剂），并在两个不同的时间提取 RNA；实验 B. 从单个供体采血并在两个不同的管中（有或没有稳定剂）运往十个实验室进行 RNA 提取。
在两种模型和采集管中，都获得了可靠的纯度、产量、GAPDH 表达和干扰结果。在没有稳定剂的管中采集的血液中，RIN（实验 A）和 IL1B、IL8 和 FOS 的转录水平（实验 B）存在很大差异。总体而言，未稳定血液样本的结果变异性更高。
我们为整个欧洲的更大环试验定义了实验设置。选择的下游分析验证了它们的潜力，可作为测试血液 RNA 质量的合适标志物。
