Published online 13 July 2006
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Genomic DNA functions as a universal external standard in quantitative real-time PCR
1 Department of Laboratory Medicine and Pathobiology, University of Toronto Toronto, Ontario, Canada M5S 1A8 2 University Health Network, Ontario Cancer Institute/Princess Margaret Hospital Toronto, Ontario, Canada M5G 2M9 3 Department of Medical Biophysics, University of Toronto Toronto, Ontario, Canada M5G 2M9 4 Centre for Research in Neurodegenerative Diseases, University of Toronto Toronto, Ontario, Canada M5S 3H2
*To whom correspondence should be addressed at Medical Science Building Room 6316, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8. Tel: +1 416 978 8878; Fax: +1 416 978 5959; Email: sandy.der{at}utoronto.ca
Received February 20, 2006. Revised May 9, 2006. Accepted May 11, 2006.
Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. E. Freitag and J. I. Prosser Correlation of Methane Production and Functional Gene Transcriptional Activity in a Peat Soil Appl. Envir. Microbiol., November 1, 2009; 75(21): 6679 - 6687. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. McQuaig, T. M. Scott, J. O. Lukasik, J. H. Paul, and V. J. Harwood Quantification of Human Polyomaviruses JC Virus and BK Virus by TaqMan Quantitative PCR and Comparison to Other Water Quality Indicators in Water and Fecal Samples Appl. Envir. Microbiol., June 1, 2009; 75(11): 3379 - 3388. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Yue, T. A. Ponzio, R. L. Fields, and H. Gainer Oxytocin and vasopressin gene expression and RNA splicing patterns in the rat supraoptic nucleus Physiol Genomics, November 12, 2008; 35(3): 231 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. N. Patel, J. Berghout, F. E. Lovegrove, K. Ayi, A. Conroy, L. Serghides, G. Min-oo, D. C. Gowda, J. V. Sarma, D. Rittirsch, et al. C5 deficiency and C5a or C5aR blockade protects against cerebral malaria J. Exp. Med., May 12, 2008; 205(5): 1133 - 1143. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Lau, P. C. Boutros, M. Pintilie, F. H. Blackhall, C.-Q. Zhu, D. Strumpf, M. R. Johnston, G. Darling, S. Keshavjee, T. K. Waddell, et al. Three-Gene Prognostic Classifier for Early-Stage Non Small-Cell Lung Cancer J. Clin. Oncol., December 10, 2007; 25(35): 5562 - 5569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. E. Lovegrove, S. A. Gharib, S. N. Patel, C. A. Hawkes, K. C. Kain, and W. C. Liles Expression Microarray Analysis Implicates Apoptosis and Interferon-Responsive Mechanisms in Susceptibility to Experimental Cerebral Malaria Am. J. Pathol., December 1, 2007; 171(6): 1894 - 1903. [Abstract] [Full Text] [PDF]
|
|