Multidimensional differential display via ion-pair reversed-phase denaturing high-performance liquid chromatography.
Multidimensional differential display via ion-pair reversed-phase denaturing high-performance liquid chromatography.
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通过离子对反相变性高效液相色谱进行多维差异显示。
DOI：
10.1006/abio.2001.5563
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发表时间：
2002
影响因子：
2.9
通讯作者：
D. Hornby
中科院分区：
文献类型：
--
作者：
M. Matin;P. Andrews;D. Hornby
实验方法现在可用于分析细胞和组织中的全转录组表达。自从引入此类方法来研究mRNA群体的差异以来，它们已成功应用于生物学和医学的许多领域，包括发育、分化、生理学、药理学和致癌作用。在这里，我们描述了一种基于Liang和Pardee（P. Liang和A. B。Pardee，1992，Science 257，967-971）。我们报告使用离子对反相变性高效液相色谱法（IP RP DHPLC），第一次，产生一个“指纹”，扩增后的cDNA对应的mRNA群体，从两个或两个以上的样品进行比较。通过重叠由来自同一组寡脱氧核苷酸引物的不同样品的扩增产生的色谱图，可以选择那些差异表达的基因，随后快速克隆和测序以建立差异表达基因的谱。此外，通过使用IP RP DHPLC和定量RT-PCR的该方法容易地实现所获得的数据的验证。在这项研究中，总RNA制备从NTERA 2细胞分化前和分化后的视黄酸诱导，并逆转录成cDNA扩增之前，产生荧光标记的产品。这种方法便于RT-PCR产物的多轮询问，我们暂时将这种方法称为多维差异显示。
Experimental approaches are now available for the analysis of whole transcriptome expression in cells and tissues. Since the introduction of such methods for the investigation of differences in mRNA populations, they have been applied successfully to many areas of biology and medicine including development, differentiation, physiology, pharmacology, and carcinogenesis. Here we describe an improved and automated approach based on the differential mRNA display method developed by Liang and Pardee (P. Liang and A. B. Pardee, 1992, Science 257, 967-971). We report the use of ion-pair reversed-phase denaturing high-performance liquid chromatography (IP RP DHPLC), for the first time, to produce a "fingerprint," after amplification of the cDNA corresponding to the mRNA populations, from two or more of the samples that are to be compared. By overlaying the chromatograms produced from the amplification of different samples derived from the same set of oligodeoxynucleotide primers, those genes that are differentially expressed can be selected and subsequently cloned and sequenced rapidly to establish a profile of differentially expressed genes. In addition, validation of the data obtained is readily achieved by this method using IP RP DHPLC and quantitative RT-PCR. In this study total RNA was prepared from NTERA2 cells before and after differentiation induced by retinoic acid and was reverse-transcribed into cDNA prior to amplification to produce fluorescently tagged products. This methodology facilitates multiple rounds of interrogation of RT-PCR products and we tentatively refer to this approach as Multidimensional Differential Display.
影响因子：
2.7
作者：
ANDREWS, PW
通讯作者：
ANDREWS, PW
DOI：
--
发表时间：
1984-02
期刊：
Laboratory investigation; a journal of technical methods and pathology
影响因子：
--
作者：
P. Andrews;I. Damjanov;D. Simon;G. Banting;C. Carlin;N. Dracopoli;J. Fogh
通讯作者：
P. Andrews;I. Damjanov;D. Simon;G. Banting;C. Carlin;N. Dracopoli;J. Fogh
从有限数量的 RNA 中鉴定差异表达基因。
影响因子：
14.9
作者：
Bosch,I;Melichar,H;Pardee,AB
通讯作者：
Pardee,AB