Reference gene validation for the relative quantification of cannabinoid receptor expression in human odontoblasts via quantitative polymerase chain reaction

Autores organización

• 

  Deisy Julieth Abril Riaño

  Autor

• 

  Jaime Eduardo Castellanos Parra

  Autor

Autores

• Navarro-Saiz LM
• Bernal-Cepeda LJ
• García-Jiménez F

Grupos de investigación

• Grupo de Virología

  

  Investigador

  Jaime Eduardo Castellanos Parra

• Laboratorio de Genética Molecular Bacteriana LGMB

  

  Investigador

  Javier Antonio Escobar Perez

Resumen

Objective: The aim of this study was to identify and validate the reference genes in cultured human odontoblasts to quantify their cannabinoid receptor transcripts. Methods: The most stably transcribed genes in cultured human odontoblast cells were identified using the RefGenes tool and were selected for real-time polymerase chain reaction (PCR) amplification. Human odontoblast cells were differentiated from mesenchymal stem cells using a transforming growth factor-ß-supplemented differentiation medium, and total RNA was purified. Reverse transcription-quantitative PCR and relative quantification analyses were performed using the Schefe's method. The relative expression dataset was analyzed to select the most stable genes. Results: The analysis showed that the transcripts of cholinergic receptor nicotinic beta 2 subunit, LIM homeobox transcription factor 1 beta, and family with sequence similarity 223 member B presented the lowest standard deviation (SD) in expression (SD: 0.2, 0.17, and 0.16, respectively). These genes showed similar expression levels as the target genes (cannabinoid receptors). Significant differences were found in the relative expression levels of cannabinoid receptors using the selected genes compared to those calculated using beta actin transcripts as references (p < 0.05). Conclusions: The strategy reported here for searching and verifying new reference genes will aid in the accurate and reliable expression of cannabinoid receptors in human odontoblast cells. © 2022

© 2022 The Authors. Published by Elsevier B.V. on behalf of Craniofacial Research Foundation.

Keywords

• Cannabinoid receptor 1; Cannabinoid receptor 2; Odontoblast; Reference gene; RT-qPCR