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Gene expression measurements in the context of epidemiological studies.

机译：流行病学研究中的基因表达测量。

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Background: Gene expression measurements became an attractive tool to assess biological responses in epidemiological studies. However, collection of blood samples poses various technical problems. We used gene expression data from two epidemiological studies to evaluate differences between sampling methods, comparability of two methods for measuring RNA levels and stability of RNA samples over time. Methods: For the PARSIFAL study, PBLC of 1155 children were collected using EDTA tubes in two countries. In the PASTURE study, tubes containing RNA-stabilizing solutions (PAXgene((R)) Blood RNA Tubes; PreAnalytiX) were used to collect cord blood leucocytes of 982 children in five countries. Real-time PCR (conventional single tube assay and high-throughput low density arrays) was used to quantify expression of various innate immunity genes. In 77 PARSIFAL samples, gene expression was measured repeatedly during prolonged storage. Results: In PARSIFAL (EDTA tubes) the median RNA yield after extraction significantly differed between the two centres (70 and 34 ng/mul). Collecting blood into an RNA-stabilizing solution markedly reduced differences in RNA yield in PASTURE (range of medians 91-107 ng/mul). The agreement [Spearman rank correlation (r)] between repeated measurements of gene expression decreased with increasing storage time [e.g., for CD14: r (first/second measurement) = 0.35; r (first/third measurement) = 0.03]. RNA levels measured with either the conventional method or low-density arrays were comparable (r > 0.9). Conclusion: Collecting blood samples into tubes containing an RNA-stabilizing solution increases RNA yield and reduces its variability. Long-term storage of samples may lead to RNA degradation, requiring special attention in longitudinal studies.
机译：背景：基因表达测量成为流行病学研究中评估生物学反应的一种有吸引力的工具。然而，血液样本的采集带来了各种技术问题。我们使用来自两项流行病学研究的基因表达数据来评估采样方法之间的差异，两种用于测量RNA水平的方法的可比性以及随时间推移RNA样品的稳定性。方法：为进行PARSIFAL研究，在两个国家中使用EDTA管收集了1155名儿童的PBLC。在PASTURE研究中，装有RNA稳定溶液的试管（PAXgene（R）Blood RNA Tubes; PreAnalytiX）被用于收集五个国家的982名儿童的脐带血白细胞。实时PCR（常规单管测定和高通量低密度阵列）用于定量各种先天免疫基因的表达。在77个PARSIFAL样品中，长时间存储过程中反复测量了基因表达。结果：在PARSIFAL（EDTA管）中，两个中心提取后的中值RNA产量显着不同（70和34 ng / mul）。将血液收集到稳定的RNA溶液中可显着降低PASTURE中RNA产量的差异（中位数范围为91-107 ng / mul）。重复测量基因表达之间的一致性[斯皮尔曼等级相关性（r）]随保存时间的增加而降低[例如，对于CD14：r（第一次/第二次测量）= 0.35; r（第一/第三次测量）= 0.03]。使用常规方法或低密度阵列测量的RNA水平具有可比性（r> 0.9）。结论：将血液样本收集到装有RNA稳定溶液的试管中，可以增加RNA产量并降低其变异性。样品的长期储存可能导致RNA降解，在纵向研究中需要特别注意。

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