Rapid quantification of murine ABC mRNAs by real time reverse transcriptase-polymerase chain reaction

Yan Ru Su,Macrae F Linton,Sergio Fazio

doi:10.1194/jlr.d200020-jlr200

Abstract

Several ATP-binding cassette (ABC) transporters are critically involved in cholesterol and phospholipid efflux, reverse cholesterol transport, and play an important role in the development of atherosclerosis. Quantification of ABC mRNA is important in studying the regulation of cellular cholesterol homeostasis and mechanisms related to the pathogenesis of atherosclerosis. We have developed a one-step real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for measuring mRNA levels of ABCA1, ABCG1, and ABCA2 in murine tissues using the TaqMan(TM) technology. It has significant methodological benefits when compared to classic Northern blotting or semi-quantitative RT-PCR analysis. Using this method, we found high expression levels of ABCA1 in liver and macrophages, and of ABCG1 in the brain and macrophages. The expression of ABCA1 and ABCG1 were further induced in macrophages loaded with acLDL. In contrast, ABCA2 was expressed exclusively in the brain with low expression levels in the macrophages. This method provides a rapid, highly sensitive, specific, and reproducible quantification of ABC mRNA, and can be performed with nanograms of total RNA sample, thus making it a superior method for studying the regulation of ABC transporters in cholesterol efflux and its role in the pathogenesis of atherosclerosis in murine models.

Highlights

Several ATP-binding cassette (ABC) transporters are critically involved in cholesterol and phospholipid efflux, reverse cholesterol transport, and play an important role in the development of atherosclerosis
Quantification of mRNA levels is often required in studying mammalian gene expression
Several methods can be used for quantitative measurement of mRNA of a target gene, such as Northern blotting analysis, RNAse protection assay, in situ hybridization, and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)

Summary

Introduction

Several ATP-binding cassette (ABC) transporters are critically involved in cholesterol and phospholipid efflux, reverse cholesterol transport, and play an important role in the development of atherosclerosis. We have developed a one-step real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for measuring mRNA levels of ABCA1, ABCG1, and ABCA2 in murine tissues using the TaqManTM technology. It has significant methodological benefits when compared to classic Northern blotting or semi-quantitative RT-PCR analysis. ABCA2 was expressed exclusively in the brain with low expression levels in the macrophages This method provides a rapid, highly sensitive, specific, and reproducible quantification of ABC mRNA, and can be performed with nanograms of total RNA sample, making it a superior method for studying the regulation of ABC transporters in cholesterol efflux and its role in the pathogenesis of atherosclerosis in murine models.—Su, Y.

Methods

Results

Conclusion