A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS
A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS
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DOI：
10.1073/pnas.0630494100
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发表时间：
2003-03-18
影响因子：
11.1
通讯作者：
Cantor, CR
中科院分区：
文献类型：
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作者：
Ding, CM;Cantor, CR
我们在此报告了一种结合竞争性PCR和基质辅助激光解吸电离飞行时间ms的基因表达分析方法。PCR前将标准品加入到逆转录产物中。随后，在单核苷酸多态性位点进行碱基延伸反应，并用基质辅助激光解吸电离飞行时间质谱法对产物进行定量，该方法能够相对定量和绝对定量基因表达；它是极其敏感的（只有5份DNA被量化）和高度可重复性。它还能够同时定量杂合子和选择性剪接基因的等位基因。我们将该技术与均匀质量扩展系统（Sequenom）相结合，创建了一个高通量、自动化的基因表达分析平台，每天可以准确定量来自20-500个不同样品的数百个基因。
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.