TY - JOUR
T1 - Adaptor-tagged competitive polymerase chain reaction
T2 - Amplification bias and quantified gene expression levels
AU - Kita-Matsuo, Hiroko
AU - Yukinawa, Naoto
AU - Matoba, Ryo
AU - Saito, Sakae
AU - Oba, Shigeyuki
AU - Ishii, Shin
AU - Kato, Kikuya
N1 - Funding Information:
We thank David C. Rubinsztein (CIMR, Cambridge University) for providing the total RNA samples from PC12 cells and Shoko Kawamoto for providing technical advice and useful discussions. We also thank Noriko Toshiro, Keiko Ikenaga, Satoko Kinjyo, and Mihoko Yoshino for providing technical assistance and Kenta Mitsuhara for annotating 4006 EST sequences. This work was supported by a grant-in-aid for scientific research in the priority area “genome science” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
PY - 2005/4/1
Y1 - 2005/4/1
N2 - 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.
AB - 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.
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U2 - 10.1016/j.ab.2004.11.014
DO - 10.1016/j.ab.2004.11.014
M3 - Article
C2 - 15766705
AN - SCOPUS:14844295484
SN - 0003-2697
VL - 339
SP - 15
EP - 28
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -