Cytotechnology

, Volume 62, Issue 2, pp 109–120

Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage

  • Felipe Garcia Quiroz
  • Olga M. Posada
  • Daniel Gallego-Perez
  • Natalia Higuita-Castro
  • Carlos Sarassa
  • Derek J. Hansford
  • Piedad Agudelo-Florez
  • Luis E. López
Original Research

DOI: 10.1007/s10616-010-9265-1

Cite this article as:
Quiroz, F.G., Posada, O.M., Gallego-Perez, D. et al. Cytotechnology (2010) 62: 109. doi:10.1007/s10616-010-9265-1

Abstract

Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation. This work aimed to study the stability of the housekeeping genes β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A) during the osteogenic differentiation of hBMSCs. Their stability was evaluated via RT-qPCR in 14 and 20 day differentiation assays to the osteogenic lineage. Different normalization strategies were evaluated to quantify the osteogenic markers collagen type I, bone sialoprotein and osteonectin. Cell differentiation was confirmed via alizarin red staining. The results demonstrated up-regulation of β-actin with maximum fold changes (MFC) of 4.38. GAPDH and RPL13A were not regulated by osteogenic media after 14 days and presented average fold changes lower than 2 in 20 day cultures. RPL13A (MFC < 2) had a greater stability when normalizing as a function of culture time compared with GAPDH (MFC ≤ 2.2), which resulted in expression patterns of the osteogenic markers more consistent with the observed differentiation process. The results suggest that β-actin regulation could be associated with the morphological changes characteristic of hBMSCs osteogenic differentiation, and provide evidence for the superior performance of RPL13A as a normalizer gene in osteogenic differentiation studies of hBMSCs. This work highlights the importance of validating the normalizer genes used for stem cells characterization via RT-qPCR.

Keywords

Mesenchymal stem cellsOsteogenic differentiationNormalizationGene expressionQuantitative RT-PCR

Abbreviations

RT-qPCR

Quantitative reverse transcription PCR

hBMSCs

Human bone marrow mesenchymal stem cells

ACTB

β-actin

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

RPL13A

Ribosomal protein L13A

CI

Collagen type I

ON

Osteonectin

BSP

Bone sialoprotein

NO

Non-osteogenic medium

OM

Osteogenic medium

ARS

Alizarin red staining

AFC

Average fold changes from the mean threshold cycle

MFC

Average maximum fold changes

CT

Threshold cycle

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Felipe Garcia Quiroz
    • 1
    • 5
  • Olga M. Posada
    • 1
  • Daniel Gallego-Perez
    • 2
  • Natalia Higuita-Castro
    • 1
    • 2
  • Carlos Sarassa
    • 3
  • Derek J. Hansford
    • 2
  • Piedad Agudelo-Florez
    • 4
  • Luis E. López
    • 1
  1. 1.Grupo de Investigación en Ingeniería Biomédica EIA-CES (GIBEC), Escuela de Ingeniería de AntioquiaUniversidad CESEnvigado-MedellinColombia
  2. 2.Department of Biomedical Engineering, Nanotech West LaboratoryThe Ohio State UniversityColumbusUSA
  3. 3.Orthopedics DepartmentHospital Pablo Tobón UribeMedellinColombia
  4. 4.Universidad CES - Instituto Colombiano de Medicina Tropical (ICMT)SabanetaColombia
  5. 5.DurhamUSA