Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood
Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood
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DOI：
10.1016/s0022-1759(97)00188-9
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发表时间：
1997-12-29
影响因子：
2.2
通讯作者：
Rieckmann, P
中科院分区：
文献类型：
--
作者：
Kruse, N;Pette, M;Rieckmann, P
为了促进血细胞中细胞因子 mRNA 的检测，我们开发了一种高度重复且易于执行的 RNA 分离方法，用于全血。将先前冷冻的人全血样本在硫氰酸胍溶液中裂解以分离总RNA。逆转录后，采用 PCR 方法检测 β-肌动蛋白和细胞因子 mRNA 表达（白细胞介素 (IL)2、IL4、IL10、肿瘤坏死因子 α (TNF α) 和干扰素 γ (IFN γ)），通过琼脂糖凝胶电泳确认 cDNA 的存在，并使用序列特异性荧光染料标记的内部寡核苷酸探针进行在线定量。该定量方法基于荧光染料标记探针的裂解。 PCR 过程中 Taq DNA 聚合酶的 5'-->3' 核酸内切酶活性以及通过序列检测器系统测量荧光强度 所产生的信号与超过 6 个对数浓度的样品中靶分子的起始拷贝数成正比，并且与 β-肌动蛋白或细胞因子 cDNA 标准品相比，可以在从 8 名健康供体的全血制备的 cDNA 中轻松检测到编码 β-肌动蛋白和 TNF α 的 mRNA。细胞因子的表达量较低（IFN γ、IL10）或无法检测到（IL2、IL4）。所描述的测定具有高度重复性，不需要对扩增子进行 PCR 后操作，并且允许每天分析数百个 PCR 反应，即使在样品储存至少几个月后，也可以可靠地检测和定量少量先前冷冻的血液中的细胞因子 mRNA 表达。
In order to facilitate cytokine mRNA detection in blood cells, we have developed a highly reproducible and easily performed RNA isolation method for use with whole blood. Previously frozen human whole blood samples were lysed in guanidine thiocyanate solution to isolate total RNA. After reverse transcription a PCR method was applied to detect beta-actin and cytokine mRNA expression (interleukin-(IL)2, IL4, IL10, tumor necrosis factor alpha (TNF alpha) and interferon gamma (lFN gamma), The presence of cDNA was confirmed by agarose gel electrophoresis and quantitated on-line using sequence-specific fluorochrome labeled internal oligonucleotide probes. This quantitative method is based on the cleavage of fluorescent dye labeled probes by the 5'-->3' endonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detector System. The signal generated was directly proportional to the starting copy number of target molecules in the sample over 6 log concentrations and quantitative analysis of cDNA concentrations was performed in comparison to beta-actin or cytokine cDNA standards. mRNAs coding for beta-actin and TNF alpha were readily detectable in cDNAs prepared from the whole blood of eight healthy donors, while the other cytokines were expressed in lower amounts (IFN gamma, IL10) or were undetectable (IL2, IL4). The assay described is highly reproducible, requires no post PCR manipulation of the amplicons and permits the analysis of several hundred PCR reactions per day. Using this method it is possible to detect and quantify cytokine mRNA expression reliably in small amounts of previously frozen blood even after storage of samples for at least several months. (C) 1997 Elsevier Science B.V.