Adamski Mateusz G, Li Yan, Wagner Erin, Yu Hua, Seales-Bailey Chloe, Soper Steven A, Murphy Michael, Baird Alison E
1Department of Neurology, SUNY Downstate Medical Center, Brooklyn, NY, USA.
J Biomol Screen. 2013 Oct;18(9):1008-17. doi: 10.1177/1087057113489882. Epub 2013 May 20.
Clinical studies of gene expression are increasingly using the whole blood, peripheral blood mononuclear cells, and leukocyte subsets involved in the innate and adaptive immune responses. However, the small amount of RNA available in the clinical setting is a limitation for commonly used methods such as quantitative polymerase chain reactions (qPCR) and microarrays. Our aim was to design 96 gene assays to simultaneously measure gene expression in the whole blood and seven leukocyte subsets using a new-generation qPCR method--high-throughput nanofluidic reverse transcription qPCR (HT RT-qPCR). The leukocyte subset purity was 94% to 98% for seven subsets and was less for the γδ T-cell receptor subset (80%). The HT RT-qPCR replicate sample measurements were highly reproducible (r = 0.997, p < 2.2 × 10(-16)), and the ΔΔCt values from HT RT-qPCR correlated significantly with those from qPCR. The control genes were differentially expressed across the eight leukocyte subsets in the control subjects (p = 1.3 × 10(-5), analysis of variance). Two analytical methods, absolute and relative, gave concordant results and were significantly correlated (p = 1.9 × 10(-9)). HT RT-qPCR permits the rapid, reproducible, and quantitative measurement of multiple transcripts using minimal sample amounts. The protocol described yielded leukocyte subsets of high purity and identified two analytic methods for use.
基因表达的临床研究越来越多地使用全血、外周血单个核细胞以及参与固有免疫和适应性免疫反应的白细胞亚群。然而，临床环境中可获得的RNA量较少，这对定量聚合酶链反应（qPCR）和微阵列等常用方法来说是一个限制。我们的目标是设计一种96基因检测方法，使用新一代qPCR方法——高通量纳米流体逆转录qPCR（HT RT-qPCR），同时测量全血和七个白细胞亚群中的基因表达。七个亚群的白细胞亚群纯度为94%至98%，而γδT细胞受体亚群的纯度较低（80%）。HT RT-qPCR重复样本测量具有高度可重复性（r = 0.997，p < 2.2×10⁻¹⁶），并且HT RT-qPCR的ΔΔCt值与qPCR的ΔΔCt值显著相关。在对照受试者的八个白细胞亚群中，对照基因存在差异表达（p = 1.3×10⁻⁵，方差分析）。绝对和相对两种分析方法得出了一致的结果，并且显著相关（p = 1.9×10⁻⁹）。HT RT-qPCR允许使用最少的样本量快速、可重复且定量地测量多个转录本。所描述的方案产生了高纯度的白细胞亚群，并确定了两种可供使用的分析方法。
