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Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages.

Lee WJ, Jeon RH, Jang SJ, Park JS, Lee SC, Baregundi Subbarao R, Lee SL, Park BW, King WA, Rho GJ - Stem Cells Int (2015)

Bottom Line: In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings.Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt.Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Republic of Korea ; PWG Genetics Pte Ltd., 15 Tech Park Crescent, Singapore 638117.

ABSTRACT
The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.

No MeSH data available.


Related in: MedlinePlus

Morphology of MSCs and cell surface antigen expression. BMSCs, AMSCs, and SMSCs were cultured in separate 35 mm dishes and displayed the morphology at 4 passages in phase contrast images (a). Three kinds of MSCs were analyzed for MSCs specific surface antigen expression by flow cytometry (b). Open histograms imply isotype IgG expression as control, and filled histograms represent each surface antigen expression. Mean% ± SD of each MSCs expression in respective three biological replications was presented on top of each graph. BMSCs: bone marrow derived mesenchymal stem cells; AMSCs: adipose derived mesenchymal stem cells; SMSCs: skin derived mesenchymal stem cells. Magnification ×100.
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fig1: Morphology of MSCs and cell surface antigen expression. BMSCs, AMSCs, and SMSCs were cultured in separate 35 mm dishes and displayed the morphology at 4 passages in phase contrast images (a). Three kinds of MSCs were analyzed for MSCs specific surface antigen expression by flow cytometry (b). Open histograms imply isotype IgG expression as control, and filled histograms represent each surface antigen expression. Mean% ± SD of each MSCs expression in respective three biological replications was presented on top of each graph. BMSCs: bone marrow derived mesenchymal stem cells; AMSCs: adipose derived mesenchymal stem cells; SMSCs: skin derived mesenchymal stem cells. Magnification ×100.

Mentions: MSCs isolated from bone marrow, subcutaneous adipose, and dermal tissues were observed to have similar fibroblastic morphologies with dendritic spindle shapes and grew as adherent cells in culture dishes (Figure 1(a)). MSCs from all tissue types expressed MSCs specific markers, such as CD29, CD44, CD90, and vimentin, whereas CD45, a hematopoietic stem cell marker, was negative in all MSCs as determined by FACS analysis as shown in Figure 1(b).


Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages.

Lee WJ, Jeon RH, Jang SJ, Park JS, Lee SC, Baregundi Subbarao R, Lee SL, Park BW, King WA, Rho GJ - Stem Cells Int (2015)

Morphology of MSCs and cell surface antigen expression. BMSCs, AMSCs, and SMSCs were cultured in separate 35 mm dishes and displayed the morphology at 4 passages in phase contrast images (a). Three kinds of MSCs were analyzed for MSCs specific surface antigen expression by flow cytometry (b). Open histograms imply isotype IgG expression as control, and filled histograms represent each surface antigen expression. Mean% ± SD of each MSCs expression in respective three biological replications was presented on top of each graph. BMSCs: bone marrow derived mesenchymal stem cells; AMSCs: adipose derived mesenchymal stem cells; SMSCs: skin derived mesenchymal stem cells. Magnification ×100.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4417979&req=5

fig1: Morphology of MSCs and cell surface antigen expression. BMSCs, AMSCs, and SMSCs were cultured in separate 35 mm dishes and displayed the morphology at 4 passages in phase contrast images (a). Three kinds of MSCs were analyzed for MSCs specific surface antigen expression by flow cytometry (b). Open histograms imply isotype IgG expression as control, and filled histograms represent each surface antigen expression. Mean% ± SD of each MSCs expression in respective three biological replications was presented on top of each graph. BMSCs: bone marrow derived mesenchymal stem cells; AMSCs: adipose derived mesenchymal stem cells; SMSCs: skin derived mesenchymal stem cells. Magnification ×100.
Mentions: MSCs isolated from bone marrow, subcutaneous adipose, and dermal tissues were observed to have similar fibroblastic morphologies with dendritic spindle shapes and grew as adherent cells in culture dishes (Figure 1(a)). MSCs from all tissue types expressed MSCs specific markers, such as CD29, CD44, CD90, and vimentin, whereas CD45, a hematopoietic stem cell marker, was negative in all MSCs as determined by FACS analysis as shown in Figure 1(b).

Bottom Line: In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings.Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt.Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Republic of Korea ; PWG Genetics Pte Ltd., 15 Tech Park Crescent, Singapore 638117.

ABSTRACT
The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.

No MeSH data available.


Related in: MedlinePlus