Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood
Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood
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DOI：
10.1373/clinchem.2005.065078
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发表时间：
2006-07-01
影响因子：
9.3
通讯作者：
McMaster, GK
中科院分区：
文献类型：
--
作者：
Zheng, Z;Luo, YL;McMaster, GK
背景：血液处理过程中基因表达的变化以及样品制备引入的变化和偏差使得全血中 mRNA 的准确定量变得困难。我们寻求开发一种定量全血 mRNA 测定，消除血液纯化、RNA 分离、逆转录和靶标扩增，同时以简单的测定形式提供高质量数据。方法：我们使用多个杂交探针进行单基因和多重基因表达分析，直接从血液裂解物中捕获 mRNA，并使用分支 DNA 放大信号。 96 孔板单重检测采用化学发光检测，而多重检测将 Luminex 编码珠与荧光检测相结合：结果：单重检测和多重检测可分别定量测量最多 25 μL 全血中的 6000 个和 24 000 个 mRNA 靶分子（0.01 和 0.04 阿摩尔）。两种格式的 CV < 10% 和 3-4 个对数的动态范围。检测灵敏度允许定量测量全血中少数细胞的基因表达。全血裂解物的信号与同一样品的纯化 RNA 的信号密切相关，并且测定的绝对 mRNA 定量结果相似。通过定量反向转录-PCR 获得的结果。单次和多重检测形式均与常用抗凝剂和 PAX 基因处理的样品兼容；然而，PAXgene 制剂可诱导全血中已知抗凋亡基因的表达。结论：单重和多重分支 DNA 测定均可直接定量测量小体积全血中的 mRNA 表达。该检测为当前依赖 RNA 分离的技术提供了一种替代方案，并且适用于全血的高通量基因表达分析。 (c) 2006 年美国临床化学协会。
Background: Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format.Methods: We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection:Results: The single- and multiplex assays could quantitatively measure as few as 6000 and 24 000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 mu L of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar. to those obtained by quantitative reverse, transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAX-gene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood.Conclusions: Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood. (c) 2006 American Association for Clinical Chemistry.