Reference Gene U2 Enables Direct Comparison between Relative Gene Expression Levels of Vascular Smooth Muscle Cells in Tissue and Culture Using Real-Time Quantitative PCR

Christine Rager; Tobias Klöpper; Uwe Pfeil; Sabine Tasch; Michael Raymond Whittaker; Betty Exintaris; Andrea Mietens; Ralf Middendorff

doi:10.3390/cells12172135

Reference Gene U2 Enables Direct Comparison between Relative Gene Expression Levels of Vascular Smooth Muscle Cells in Tissue and Culture Using Real-Time Quantitative PCR

Cells. 2023 Aug 23;12(17):2135. doi: 10.3390/cells12172135.

Authors

Christine Rager^{1

2}, Tobias Klöpper¹, Uwe Pfeil³, Sabine Tasch¹, Michael Raymond Whittaker², Betty Exintaris⁴, Andrea Mietens¹, Ralf Middendorff¹

Affiliations

¹ Signalling Transduction Group, Institute of Anatomy and Cell Biology, Faculty of Medicine, Justus-Liebig-University, 35392 Giessen, Germany.
² Drug Discovery Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Melbourne, VIC 3052, Australia.
³ Cardiopulmonary Neurobiology Group, Institute of Anatomy and Cell Biology, Faculty of Medicine, Justus-Liebig-University, 35392 Giessen, Germany.
⁴ Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Melbourne, VIC 3052, Australia.

Abstract

In nearly every lab, real-time quantitative polymerase chain reaction (qPCR) is used to quantify gene expression. However, a comparison of different samples requires the careful selection of suitable reference genes (RGs), sometimes referred to as housekeeping genes. In the case of vascular smooth muscle cells (vSMCs), it is important to know under which conditions gene expression in isolated and cultured vSMCs can be compared with vSMCs in a healthy blood vessel. We isolated the vSMC-containing layer of the rat aorta (tunica media) and used one (longitudinal) half for direct RNA extraction, while the other half served to isolate and culture vSMCs prior to RNA extraction. First, the expression of the routinely used RGs beta-actin (Actb) and Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is investigated in intact media and corresponding cultured vSMCs. Significant differences in their Ct values show that these RGs could not be used for such direct comparisons; therefore, we select 15 different RGs. Only the gene expression of the small ribonuclear protein (snRNP) U2 shows no significant differences between the absolute Ct values of cultured vSMCs and the intact media; moreover, no differences were found between male and female rats in our experimental setup. In conclusion, U2 was shown to be an appropriate (sex-independent) RG to compare relative expression levels of vSMCs in culture to those vSMCs within their physiological tissue environment.

Keywords: aorta; qPCR; reference gene (housekeeping gene); vascular smooth muscle cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Female
Gene Expression
Genes, Essential*
Male
Muscle, Smooth, Vascular*
RNA
Rats
Real-Time Polymerase Chain Reaction

Substances

RNA

Grants and funding

This work was funded by the Deutsche Forschungsgesellschaft (DFG) as part of the International Research Training Group (IRTG) 1871 (Grant/Award Number GRK 1871) and by the Hessian Ministry of Science and Art in the research category “Frauen- und Geschlechterforschung in Hessen”/“Women- and Gender research in Hesse”. As member of the IRTG 1871, C.R. received a Monash Graduate Scholarship (MGS) of the Faculty for Medicine, Nursing and Health Sciences at Monash University, Melbourne, VIC, Australia.