Determination of murine fetal Cyp1a1 and 1b1 expression by real-time fluorescence reverse transcription-polymerase chain reaction

Abstract

We describe the application of quantitative RT-PCR methodology to study expression of Cyp1a1 and 1b1 in murine fetal tissue. We applied real-time PCR using the fluorescent dye, SYBR Green, to establish a very reliable, sensitive, specific, inexpensive, and rapid assay that was linear over a wide dynamic range of starting target gene copy number. The main point of this paper is to provide a detailed description of how to optimize real-time reverse transcription-polymerase chain reaction (RT-PCR) assays and, most importantly, to provide solutions for some of the commonly observed problems encountered when employing quantitative RT-PCR methods. We establish that use of SYBR Green for detection of PCR products was a powerful and reliable tool in our investigation of Cyp gene expression in murine fetal tissues, and demonstrate the kinetics of induction of Cyp1a1 and 1b1 in crosses between C57BL/6 and Balb/c mice. In addition, we demonstrate for the first time the induction of Cyp1b1 RNA expression in fetal lung and liver tissues following in utero exposure to 3-methylcholanthrene (MC), a polycyclic aromatic hydrocarbon. This assay for Cyp1a1 and 1b1 expression could be utilized equally well to detect other genes of interest in a variety of toxicological investigations.

Introduction

Gene expression analysis plays an increasingly important role in many fields of toxicology. Precise quantification of mRNA expression by conventional methods, such as Northern blotting and ribonuclease protection assays, require large amounts of RNA, and are not always feasible when the transcripts of interest display low expression levels and/or sample size may be limiting. Conventional reverse transcription-polymerase chain reaction (RT-PCR) can overcome some of these limitations but requires elaborate and very time-consuming standardization protocols and post-PCR manipulations, making high-throughput analyses difficult. Over the past decade, several investigators have reported the development of real-time PCR quantification methods that have many advantages over conventional RT-PCR in terms of accuracy, sensitivity, dynamic range, high-throughput capacity, and absence of post-PCR manipulation. In these approaches, sequence-specific fluorescence-labeled probes or primers (e.g. TaqMan, Molecular Beacons, and Scorpions) have been considered as detection formats in many research applications Gut et al., 1999, Thelwell et al., 2000, Tyagi and Kramer, 1996 and have greatly enhanced our ability to measure and accurately quantify low copy number transcripts in very small samples. One drawback to these methods is that relatively expensive probes, primers, or both are required for each amplicon under investigation.

A low-cost alternative to these systems is the SYBR Green I DNA-binding dye method, which has several advantages and disadvantages compared to methods utilizing fluorescent primers or probes (Morrison et al., 1998). SYBR Green I is a DNA-binding dye that incorporates nondiscriminately into dsDNA. It has an undetectable fluorescence when it is in its free form but, once bound to dsDNA, emits a detectable fluorescent signal that linearly increases with the formation of PCR products. The major advantage of SYBR Green I dye is that it can be used with any pair of primers for any target gene PCR product, making it easy to optimize PCR reaction conditions for several different genes and at much reduced cost compared to other probe and primer labeling methods. On the other hand, SYBR Green dye not only binds to target dsDNA but also to nonspecific PCR products and primer dimers, thus necessitating efficient primer design. In addition, among the disadvantages of SYBR Green is the fact that, because binding of the dye is not sequence-specific, it cannot be used when multplex reactions are required. However, for many applications, fluorescent detection the SYBR Green is a very reliable and cost-saving assay.

Two different methods are utilized to provide quantitative RT-PCR data. Absolute quantification requires the construction of an absolute standard curve for each individual amplicon to calculate the precise copy number of mRNA transcripts based on an accurate determination of the number of cells or unit mass of tissue (Orlando et al., 1998). Alternatively, one of the relative quantification methods can be used. In the relative standard curve method, the input amount for unknown samples is calculated from a standard curve generated from the specific gene product, which is then normalized to the input amount of a reference gene (also calculated from its own standard curve). The relative comparative ΔΔC_t method utilizes a direct comparison of the difference when samples cross the threshold for detection and is based on the assumption that the amplification efficiency of the target gene and the internal control gene are the same Livak, 1997, Livak and Schmittgen, 2001. One limitation of this approach is that the amplification efficiencies of the reference and target genes must be nearly identical, which is often difficult to achieve.

Our laboratory has been studying the role of Cyp1a1 and 1b1 in fetal tissues in determining the susceptibility of the fetus to chemically induced lung and liver tumors following in utero exposure to environmental carcinogens (reviewed in Miller, 2004, Miller et al., 2000). Quantification of Cyp gene induction during the fetal period has been determined primarily by changes in the metabolism of Cyp-selective substrates; by direct quantification of Cyp protein using specific antibodies, Western blotting, or immunohistochemical approaches; and by more common methodologies used in the determination of RNA gene expression (e.g., Northern blot, slot-blot, and RNase protection assays) following transplacental exposure to environmental toxicants (Dey et al., 1998; reviewed in Miller, 2004, Miller et al., 1990). In our own studies in this area, we have experienced several obstacles in obtaining accurate quantitation with these conventional methods, including the need for large amounts of RNA, the relatively small sample sizes and yields of RNA from small amounts of murine fetal tissues, and the laborious post-PCR manipulations (i.e., band densitometry, radioactive labeling, and phosphorimaging). Importantly, these methods are typically semiquantitative in nature.

Interestingly, when we initially employed real-time PCR quantitative methods to attempt to more accurately quantitate Cyp induction in fetal tissues, we were surprised to find a paucity of papers utilizing this approach with these widely studied and critically important drug metabolic enzymes in murine models. This prompted us, in this report, to describe the experimental procedures we employed to standardize and optimize this assay, including solutions to problems we encountered, for other investigators wanting to utilize this technology for their own systems. In the present report, we describe our experience in the development of a real-time fluorescent RT-PCR assay to determine the expression of Cyp1a1 and 1b1 in murine fetal tissues following in utero exposure to 3-methylcholanthrene (MC). We applied quantitative RT-PCR using the SYBR Green I dye and the relative standard curve method in combination with the iCycler iQ Real-Time detection system. We show here that the method we established provided very sensitive, rapid, and reproducible results, allowing quantification over a wide dynamic range of starting target gene copies that could easily be adapted for other genes of interest. The real-time assay allowed us to demonstrate the kinetics of induction of Cyp1a1 and 1b1 RNA expression in fetal lung and liver tissue following in utero exposure to MC, and demonstrate for the first time the induction of Cyp1b1 in fetal tissues.