Adaptor-tagged competitive polymerase chain reaction: Amplification bias and quantified gene expression levels

Hiroko Kita-Matsuo, Naoto Yukinawa, Ryo Matoba, Sakae Saito, Shigeyuki Oba, Shin Ishii, Kikuya Kato

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

Abstract

Adaptor-tagged competitive polymerase chain reaction (ATAC-PCR) is an advanced version of quantitative competitive PCR characterized by the addition of unique adaptors to different cDNA samples. It is currently the only quantitative PCR technique that enables large-scale gene expression analysis. Multiplex application of ATAC-PCR employs seven adaptors, two or three of which are used as controls to generate a calibration curve. The characteristics of the ATAC-PCR method for large-scale data production, including any adaptor- and gene-dependent amplification biases, were evaluated by using this method to analyze the expression of 384 mouse brain genes. Short adaptors tended to amplify at higher efficiency than did long adaptors. The population of genes with a high amplification bias increased with the use of short adaptors. Subtracting the median value of all adaptor-dependent biases could reduce this bias; the majority of genes displayed a small gene-dependent bias, which facilitated reliable quantification. We modified ATAC-PCR to estimate molecular numbers of transcripts by introducing synthetic standards. This modification demonstrated that gene expression levels in mammalian cells are varied over seven orders of magnitude.

Original languageEnglish
Pages (from-to)15-28
Number of pages14
JournalAnalytical Biochemistry
Volume339
Issue number1
DOIs
Publication statusPublished - 2005 Apr 1
Externally publishedYes

Keywords

  • ATAC-PCR
  • Gene expression profiling
  • PC12
  • RT-PCR

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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