Expression measurement of many genes simultaneously by quantitative RT-PCR using standardized mixtures of competitive templates
Expression measurement of many genes simultaneously by quantitative RT-PCR using standardized mixtures of competitive templates
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DOI：
10.1165/ajrcmb.19.1.3076
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发表时间：
1998-07-01
影响因子：
6.4
通讯作者：
DeMuth, JP
中科院分区：
文献类型：
--
作者：
Willey, JC;Crawford, EL;DeMuth, JP
通过人类基因组计划实现所有人类基因的完全测序的进展已经导致需要允许同时定量测量多个基因的表达的方法。越来越多的人认识到，多个基因的相对测量将提供更多的关于细胞病理生理学的机制信息，而不是一个接一个地测量单个基因或通过不允许直接基因间比较的方法。在这项研究中，以前描述的定量逆转录聚合酶链反应方法进行了修改，努力提供一个快速，简单的方法，用于此目的。为每个基因制备内标竞争模板（CT），并将其组合在含有相对于彼此的规定浓度的40多种基因的CT的单一溶液中。CT混合物的任何后续稀释不会改变一个CT与另一个CT的关系。由于在所有实验中使用相同的CT标准溶液或其稀释液，因此很容易比较从不同实验获得的数据。使用具有不同管家基因与靶基因比率的多种CT混合物提供了跨越迄今为止评价的基因表达范围的线性动态范围。CT储备溶液用于同时定量25个基因的表达相对于β-肌动蛋白和甘油醛-3-磷酸脱氢酶在正常和恶性支气管上皮细胞。由于CT浓度已知，因此获得了绝对信使RNA（mRNA）拷贝数和相对于管家基因mRNA的mRNA形式的数据。所描述的方法和试剂将允许使用廉价且广泛可用的设备同时快速定量测量多个基因。此外，CT标准溶液可分发给其他研究者，用于实验条件的实验室间标准化。
Progress toward complete sequencing of all human genes through the Human Genome Project has already resulted in a need for methods that allow quantitative expression measurement of multiple genes simultaneously. It is increasingly recognized that relative measurement of multiple genes will provide more mechanistic information regarding cell pathophysiology than measurement of individual genes one by one or by methods that do not allow direct intergene comparison. In this study, previously described quantitative reverse transcription-polymerase chain reaction methods were modified in an effort to provide a rapid, simple method for this purpose. Internal standard competitive templates (CTs) were prepared for each gene and were combined in a single solution containing CTs for more than 40 genes at defined concentrations relative to one another. Any subsequent dilution of the CT mixture did not alter the relationship of one CT to another. Because the same CT standard solution or a dilution of it was used in all experiments, data obtained from different experiments were easily compared. The use of multiple CT mixtures with different housekeeping gene to target gene ratios provided a linear dynamic range spanning the range of expression of an genes thus far evaluated. CT stock solutions were used to simultaneously quantify the expression of 25 genes relative to beta-actin and glyceraldehyde-3-phosphate dehydrogenase in normal and malignant bronchial epithelial cells. Because the CT concentrations were known, data in the form of both absolute messenger RNA (mRNA) copy number and mRNA relative to housekeeping gene mRNA were obtained. The methods and reagents described will allow rapid, quantitative measurement of multiple genes simultaneously, using inexpensive and widely available equipment. Furthermore, the CT standard solution may be distributed to other investigators for interlaboratory standardization of experimental conditions.