PMID- 20396946
OWN - NLM
STAT- PubMed-not-MEDLINE
DCOM- 20111110
LR  - 20211020
IS  - 0920-9069 (Print)
IS  - 1573-0778 (Electronic)
IS  - 0920-9069 (Linking)
VI  - 62
IP  - 2
DP  - 2010 Apr
TI  - Housekeeping gene stability influences the quantification of osteogenic markers 
      during stem cell differentiation to the osteogenic lineage.
PG  - 109-20
LID - 10.1007/s10616-010-9265-1 [doi]
AB  - 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 beta-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 beta-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 beta-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.
FAU - Quiroz, Felipe Garcia
AU  - Quiroz FG
AD  - Grupo de Investigacion en Ingenieria Biomedica EIA-CES (GIBEC), Escuela de 
      Ingenieria de Antioquia, Universidad CES, Envigado-Medellin, Colombia, 
      bmfegar@eia.edu.co.
FAU - Posada, Olga M
AU  - Posada OM
FAU - Gallego-Perez, Daniel
AU  - Gallego-Perez D
FAU - Higuita-Castro, Natalia
AU  - Higuita-Castro N
FAU - Sarassa, Carlos
AU  - Sarassa C
FAU - Hansford, Derek J
AU  - Hansford DJ
FAU - Agudelo-Florez, Piedad
AU  - Agudelo-Florez P
FAU - Lopez, Luis E
AU  - Lopez LE
LA  - eng
PT  - Journal Article
DEP - 20100416
PL  - United States
TA  - Cytotechnology
JT  - Cytotechnology
JID - 8807027
PMC - PMC2873986
EDAT- 2010/04/17 06:00
MHDA- 2010/04/17 06:01
PMCR- 2011/04/01
CRDT- 2010/04/17 06:00
PHST- 2009/09/30 00:00 [received]
PHST- 2010/03/18 00:00 [accepted]
PHST- 2010/04/17 06:00 [entrez]
PHST- 2010/04/17 06:00 [pubmed]
PHST- 2010/04/17 06:01 [medline]
PHST- 2011/04/01 00:00 [pmc-release]
AID - 9265 [pii]
AID - 10.1007/s10616-010-9265-1 [doi]
PST - ppublish
SO  - Cytotechnology. 2010 Apr;62(2):109-20. doi: 10.1007/s10616-010-9265-1. Epub 2010 
      Apr 16.