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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS.
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A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS.

机译：使用竞争性PCR和基质辅助激光解吸电离飞行时间MS的高通量基因表达分析技术。

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摘要

We report here an approach for gene expression analysis by combining competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. A DNA standard is designed with an artificial single nucleotide polymorphism in the gene of interest. The standard is added to the reverse transcription product before PCR. Subsequently, a base extension reaction is carried out at the single nucleotide polymorphism position, and the products are quantified by matrix-assisted laser desorption ionization time-of-flight MS. The approach is capable of relative and absolute quantification of gene expression; it is extremely sensitive (as few as five copies of DNA were quantified) and highly reproducible. It is also capable of simultaneous quantification of both alleles for heterozygotes and alternatively spliced genes. We have incorporated this technique with the homogeneous Mass Extension system (Sequenom) to create a high-throughput, automated gene expression analysis platform where a few hundred genes from 20-500 different samples can be accurately quantified per day.
机译：我们在这里报告通过结合竞争性PCR和基质辅助的激光解吸电离飞行时间质谱仪进行基因表达分析的方法。设计一种DNA标准品，在目标基因中具有人工单核苷酸多态性。在PCR之前将标准品添加到逆转录产物中。随后，在单核苷酸多态性位置进行碱基延伸反应，并通过基质辅助激光解吸电离飞行时间MS对产物进行定量。该方法能够相对和绝对定量基因表达。它非常灵敏（定量了5个DNA拷贝）并且具有很高的重现性。它也能够同时定量杂合子和剪接基因的两个等位基因。我们已将此技术与同质传质系统（Sequenom）整合在一起，以创建一个高通量的自动化基因表达分析平台，每天可以准确地定量分析20-500个不同样品中的数百个基因。

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