Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF

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Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF. / Tratwal, Josefine; Follin, Bjarke; Ekblond, Annette; Kastrup, Jens; Haack-Sørensen, Mandana.

I: B M C Molecular Biology, Bind 15, 11, 2014, s. 1-11.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

Harvard

Tratwal, J, Follin, B, Ekblond, A, Kastrup, J & Haack-Sørensen, M 2014, 'Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF', B M C Molecular Biology, bind 15, 11, s. 1-11. https://doi.org/10.1186/1471-2199-15-11

APA

Tratwal, J., Follin, B., Ekblond, A., Kastrup, J., & Haack-Sørensen, M. (2014). Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF. B M C Molecular Biology, 15, 1-11. [11]. https://doi.org/10.1186/1471-2199-15-11

Vancouver

Tratwal J, Follin B, Ekblond A, Kastrup J, Haack-Sørensen M. Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF. B M C Molecular Biology. 2014;15:1-11. 11. https://doi.org/10.1186/1471-2199-15-11

Author

Tratwal, Josefine ; Follin, Bjarke ; Ekblond, Annette ; Kastrup, Jens ; Haack-Sørensen, Mandana. / Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF. I: B M C Molecular Biology. 2014 ; Bind 15. s. 1-11.

Bibtex

@article{86612d441e34418f9c1fbd9f8db768f2,
title = "Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF",
abstract = "BACKGROUND: Human mesenchymal stromal cells from the bone marrow (BMSCs) are widely used as experimental regenerative treatment of ischemic heart disease, and the first clinical trials using adipose-derived stromal cells (ASCs) are currently being conducted. Regenerative mechanisms of BMSCs and ASCs are manifold and in vitro pretreatment of the cells with growth factors has been applied to potentially enhance these properties. When characterizing the transcriptional activity of these cellular mechanisms in vitro it is important to consider the effect of the growth factor treatment on reference genes (RGs) for the normalization of qPCR data.RESULTS: BMSCs and ASCs were stimulated with vascular endothelial growth factor A-165 (VEGF) for one week, and compared with un-stimulated cells from the same donor. The stability of nine RGs through VEGF treatment as well as the donor variation was assessed using the GenEx software with the subprograms geNorm and Normfinder.The procedure of stepwise elimination was validated by poor performance of eliminated RGs in a normalization experiment using vWF as target gene. Normfinder found the TATA box binding protein (TBP) to be the most stable single RG for both BMSCs and ASCs. The optimal number of RGs for ASCs was two, and the lowest variance for vWF normalization was found using TBP and YWHAZ. For BMSCs, the optimal number of RGs was four, while the two-RG combination producing the most similar results was TBP and YWHAZ.CONCLUSIONS: A common reference gene, TBP, was found to be the most stable standalone gene, while TBP and YWHAZ were found to be the best two-RG combination for qPCR analyses for both BMSCs and ASCs through the VEGF stimulation. The presented stepwise elimination procedure was validated, while we found the final normalization experiment to be essential.",
keywords = "Adult, Bone Marrow Cells, Female, Genes, Humans, Male, Mesenchymal Stromal Cells, Middle Aged, Polymerase Chain Reaction, TATA-Box Binding Protein, Vascular Endothelial Growth Factor A",
author = "Josefine Tratwal and Bjarke Follin and Annette Ekblond and Jens Kastrup and Mandana Haack-S{\o}rensen",
year = "2014",
doi = "10.1186/1471-2199-15-11",
language = "English",
volume = "15",
pages = "1--11",
journal = "B M C Molecular Biology",
issn = "1471-2199",
publisher = "BioMed Central Ltd.",

}

RIS

TY - JOUR

T1 - Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF

AU - Tratwal, Josefine

AU - Follin, Bjarke

AU - Ekblond, Annette

AU - Kastrup, Jens

AU - Haack-Sørensen, Mandana

PY - 2014

Y1 - 2014

N2 - BACKGROUND: Human mesenchymal stromal cells from the bone marrow (BMSCs) are widely used as experimental regenerative treatment of ischemic heart disease, and the first clinical trials using adipose-derived stromal cells (ASCs) are currently being conducted. Regenerative mechanisms of BMSCs and ASCs are manifold and in vitro pretreatment of the cells with growth factors has been applied to potentially enhance these properties. When characterizing the transcriptional activity of these cellular mechanisms in vitro it is important to consider the effect of the growth factor treatment on reference genes (RGs) for the normalization of qPCR data.RESULTS: BMSCs and ASCs were stimulated with vascular endothelial growth factor A-165 (VEGF) for one week, and compared with un-stimulated cells from the same donor. The stability of nine RGs through VEGF treatment as well as the donor variation was assessed using the GenEx software with the subprograms geNorm and Normfinder.The procedure of stepwise elimination was validated by poor performance of eliminated RGs in a normalization experiment using vWF as target gene. Normfinder found the TATA box binding protein (TBP) to be the most stable single RG for both BMSCs and ASCs. The optimal number of RGs for ASCs was two, and the lowest variance for vWF normalization was found using TBP and YWHAZ. For BMSCs, the optimal number of RGs was four, while the two-RG combination producing the most similar results was TBP and YWHAZ.CONCLUSIONS: A common reference gene, TBP, was found to be the most stable standalone gene, while TBP and YWHAZ were found to be the best two-RG combination for qPCR analyses for both BMSCs and ASCs through the VEGF stimulation. The presented stepwise elimination procedure was validated, while we found the final normalization experiment to be essential.

AB - BACKGROUND: Human mesenchymal stromal cells from the bone marrow (BMSCs) are widely used as experimental regenerative treatment of ischemic heart disease, and the first clinical trials using adipose-derived stromal cells (ASCs) are currently being conducted. Regenerative mechanisms of BMSCs and ASCs are manifold and in vitro pretreatment of the cells with growth factors has been applied to potentially enhance these properties. When characterizing the transcriptional activity of these cellular mechanisms in vitro it is important to consider the effect of the growth factor treatment on reference genes (RGs) for the normalization of qPCR data.RESULTS: BMSCs and ASCs were stimulated with vascular endothelial growth factor A-165 (VEGF) for one week, and compared with un-stimulated cells from the same donor. The stability of nine RGs through VEGF treatment as well as the donor variation was assessed using the GenEx software with the subprograms geNorm and Normfinder.The procedure of stepwise elimination was validated by poor performance of eliminated RGs in a normalization experiment using vWF as target gene. Normfinder found the TATA box binding protein (TBP) to be the most stable single RG for both BMSCs and ASCs. The optimal number of RGs for ASCs was two, and the lowest variance for vWF normalization was found using TBP and YWHAZ. For BMSCs, the optimal number of RGs was four, while the two-RG combination producing the most similar results was TBP and YWHAZ.CONCLUSIONS: A common reference gene, TBP, was found to be the most stable standalone gene, while TBP and YWHAZ were found to be the best two-RG combination for qPCR analyses for both BMSCs and ASCs through the VEGF stimulation. The presented stepwise elimination procedure was validated, while we found the final normalization experiment to be essential.

KW - Adult

KW - Bone Marrow Cells

KW - Female

KW - Genes

KW - Humans

KW - Male

KW - Mesenchymal Stromal Cells

KW - Middle Aged

KW - Polymerase Chain Reaction

KW - TATA-Box Binding Protein

KW - Vascular Endothelial Growth Factor A

U2 - 10.1186/1471-2199-15-11

DO - 10.1186/1471-2199-15-11

M3 - Journal article

VL - 15

SP - 1

EP - 11

JO - B M C Molecular Biology

T2 - B M C Molecular Biology

JF - B M C Molecular Biology

SN - 1471-2199

M1 - 11

ER -

ID: 138312806