Absolute Quantitation of Normal and ROS-Induced Patterns of Gene Expression: An In Vivo Real-Time PCR Study in Mice

Abstract

Most studies using real-time PCR are taken semiquantitatively and assume a steady level of expression forthe so-called housekeeping genes. By absolute real-time PCR we demonstrate that the transcript amounts of two of the most popular internall controls (coding GAPDH and beta-actin) fluctuate dramatically across diverse mouse or human tissues. This raises the question about the inaccuracy of these genes a squantitative references in tissue-specific mRNA profiling. Target genes chosen for absolute real-time PCR analysis are involved in DNA repair, regulation of gene expression, and oxidative stress response. Hence, they code for 8-oxoG-DNA glycosylase/AP-lyase, major AP-endonuclease, and heme oxygenase-1. Quantitations reported: i) determine mouse-to-mouse variability in basal gene expression, ii) establish organ- and embryo-associated differences in mouse, iii) compare mouse and human tissue-specific profiles, iv) examine the time course (30-240 min) expression in liver and lung of mice treated with paraquat (superoxide generator) at 30 mg kg(-1) (one half LD50 value), and v) explore the utility of absolute real-time PCR in field studies with genetically diverse mice. We conclusively establish that real-time PCR is a highly sensitive and reproducible technique for absolute quantitation of transcript levels in vivo and propose its use to quantitate gene expression modulation under mild physiological exposures and for field epidemiological studies.

Absolute mRNA levels and transcriptional regulation of the mouse testis-specific thioredoxins

“…An absolute standard curve was constructed with an external standard in the range of 10 2 to 10 9 RNA molecules. The number of mRNA molecules was calculated from the linear regression of the standard curve, as previously described [23].…”

“…Here, we used a quantitatively rigorous approach based on reverse transcription followed by real-time PCR amplification [23] to provide for the first time a valuable new information on the steady-state copy numbers of the three murine Sptrx-1, Sprtx-2 and Sptrx-3 transcripts.…”

New Methodologies for Assessing the Presence and Ecological Effects of Pesticides in Doñana National Park (SW Spain)

“…Absolute quantification (real quantification) gives the molecule copy number of each transcript in each of the samples under study using a calibration curve. Although relative quantification is much easier to perform because a calibration curve is not necessary, relative data are much less informative than absolute data (Jurado et al, 2003;Prieto-Álamo et al, 2003;Jiménez et al, 2005;RuizLaguna et al, 2005RuizLaguna et al, , 2006. The commercialization of real-time PCR equipments allows for absolute expression data with precision levels unattainable for those generated by conventional end-time PCR.…”

“…The two murine species separated more than one million years ago (Gao & Zhang, 2003) and exhibit a relatively high gene sequence homology. Consequently, molecular studies with the aboriginal species can take advantage of the laboratory species databases (Prieto-Álamo, et al, 2003;Ruiz-Laguna et al, 2005, Several technologies are available to analyze the transcriptome, the entire complement of transcripts in a cell, tissue or organism under certain conditions. Microarrays technology allows thousands of gene transcripts to be monitored simultaneously, but they are based in the complementarity of sequences between probes and tested samples.…”

Overview of Real-Time PCR Principles

“…Also, the response of human cancer to drugs has been studied (Au, Chim et al 2002;Miyoshi, Ando et al 2002;Reimer, Koczan et al 2002). Other clinically relevant applications are cytokine mRNA profiling in immune response (Hempel, Smith et al 2002;Stordeur, Poulin et al 2002) and tissue-specific gene expression analysis (Bustin 2002;Poola 2003;Prieto-Alamo, Cabrera-Luque et al 2003).…”