Genomic DNA functions as a universal external standard in quantitative real-time PCR.
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.
Yun
James J
JJ
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Heisler
Lawrence E
LE
Hwang
Irene I L
II
Wilkins
Olivia
O
Lau
Suzanne K
SK
Hyrcza
Martin
M
Jayabalasingham
Bamini
B
Jin
Jing
J
McLaurin
JoAnne
J
Tsao
Ming-Sound
MS
Der
Sandy D
SD
D000818:Animals; D004247:DNA / Q000032:analysis / Q000592:standards*; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D018628:Gene Dosage; D020869:Gene Expression Profiling; D016678:Genome; D005838:Genotype; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results
D017931:DNA Primers; D018076:DNA, Complementary; D004247:DNA