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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.


Confirmation of differentiation ability into various mesenchymal lineages. Three kinds of MSCs were differentiated into adipocytes, osteocytes, and chondrocytes for three weeks. (a) Cytochemical staining of each differentiated MSCs with Oil red O, Von Kossa, AP staining, and alcian blue. (b) Increased intensities of lineage specific gene expression before and after differentiation in each MSCs by RT-PCR. Lanes, which were displayed from the left to the right, showed 100 bp or 200 bp ladder, undifferentiated MSCs, and differentiated cells in numerical order of BMSCs, AMSCs, and SMSCs, respectively. AP2: adipocyte fatty acid binding protein; LPL: lipoprotein lipase; OPN: osteopontin; ON: osteonectin; ACAN: aggrecan; COL10A1: collagen, type X, alpha 1; BMSC-A, BMSC-O, and BMSC-C: differentiated BMSC into adipocytes, osteoblasts, and chondrocytes, respectively; AMSC-A, AMSC-O, and AMSC-C: differentiated AMSC into adipocytes, osteoblasts, and chondrocytes, respectively; SMSC-A, SMSC-O, and SMSC-C: differentiated SMSC into adipocytes, osteoblasts, and chondrocytes, respectively. Magnification ×100.
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fig2: Confirmation of differentiation ability into various mesenchymal lineages. Three kinds of MSCs were differentiated into adipocytes, osteocytes, and chondrocytes for three weeks. (a) Cytochemical staining of each differentiated MSCs with Oil red O, Von Kossa, AP staining, and alcian blue. (b) Increased intensities of lineage specific gene expression before and after differentiation in each MSCs by RT-PCR. Lanes, which were displayed from the left to the right, showed 100 bp or 200 bp ladder, undifferentiated MSCs, and differentiated cells in numerical order of BMSCs, AMSCs, and SMSCs, respectively. AP2: adipocyte fatty acid binding protein; LPL: lipoprotein lipase; OPN: osteopontin; ON: osteonectin; ACAN: aggrecan; COL10A1: collagen, type X, alpha 1; BMSC-A, BMSC-O, and BMSC-C: differentiated BMSC into adipocytes, osteoblasts, and chondrocytes, respectively; AMSC-A, AMSC-O, and AMSC-C: differentiated AMSC into adipocytes, osteoblasts, and chondrocytes, respectively; SMSC-A, SMSC-O, and SMSC-C: differentiated SMSC into adipocytes, osteoblasts, and chondrocytes, respectively. Magnification ×100.

Mentions: MSCs from all types of tissue examined were successfully differentiated into mesenchymal lineages, adipocytes, osteocytes, and chondrocytes under the specific induction conditions. Cytochemistry was performed to evaluate differentiation and revealed that MSCs from all types of tissue made steady progress towards differentiating into adipocytes, osteocytes, and chondrocytes as conformed by Oil red O, Von Kossa, and alcian blue staining, respectively. Moreover, alkaline phosphatase activities were detected in osteogenic induced MSCs (Figure 2(a)). Furthermore, RT-PCR was performed to confirm the differentiation by assessing lineage specific gene expression, and amplified products of each gene in agarose gel were displayed in Figure 2(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)

Confirmation of differentiation ability into various mesenchymal lineages. Three kinds of MSCs were differentiated into adipocytes, osteocytes, and chondrocytes for three weeks. (a) Cytochemical staining of each differentiated MSCs with Oil red O, Von Kossa, AP staining, and alcian blue. (b) Increased intensities of lineage specific gene expression before and after differentiation in each MSCs by RT-PCR. Lanes, which were displayed from the left to the right, showed 100 bp or 200 bp ladder, undifferentiated MSCs, and differentiated cells in numerical order of BMSCs, AMSCs, and SMSCs, respectively. AP2: adipocyte fatty acid binding protein; LPL: lipoprotein lipase; OPN: osteopontin; ON: osteonectin; ACAN: aggrecan; COL10A1: collagen, type X, alpha 1; BMSC-A, BMSC-O, and BMSC-C: differentiated BMSC into adipocytes, osteoblasts, and chondrocytes, respectively; AMSC-A, AMSC-O, and AMSC-C: differentiated AMSC into adipocytes, osteoblasts, and chondrocytes, respectively; SMSC-A, SMSC-O, and SMSC-C: differentiated SMSC into adipocytes, osteoblasts, and chondrocytes, respectively. Magnification ×100.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4417979&req=5

fig2: Confirmation of differentiation ability into various mesenchymal lineages. Three kinds of MSCs were differentiated into adipocytes, osteocytes, and chondrocytes for three weeks. (a) Cytochemical staining of each differentiated MSCs with Oil red O, Von Kossa, AP staining, and alcian blue. (b) Increased intensities of lineage specific gene expression before and after differentiation in each MSCs by RT-PCR. Lanes, which were displayed from the left to the right, showed 100 bp or 200 bp ladder, undifferentiated MSCs, and differentiated cells in numerical order of BMSCs, AMSCs, and SMSCs, respectively. AP2: adipocyte fatty acid binding protein; LPL: lipoprotein lipase; OPN: osteopontin; ON: osteonectin; ACAN: aggrecan; COL10A1: collagen, type X, alpha 1; BMSC-A, BMSC-O, and BMSC-C: differentiated BMSC into adipocytes, osteoblasts, and chondrocytes, respectively; AMSC-A, AMSC-O, and AMSC-C: differentiated AMSC into adipocytes, osteoblasts, and chondrocytes, respectively; SMSC-A, SMSC-O, and SMSC-C: differentiated SMSC into adipocytes, osteoblasts, and chondrocytes, respectively. Magnification ×100.
Mentions: MSCs from all types of tissue examined were successfully differentiated into mesenchymal lineages, adipocytes, osteocytes, and chondrocytes under the specific induction conditions. Cytochemistry was performed to evaluate differentiation and revealed that MSCs from all types of tissue made steady progress towards differentiating into adipocytes, osteocytes, and chondrocytes as conformed by Oil red O, Von Kossa, and alcian blue staining, respectively. Moreover, alkaline phosphatase activities were detected in osteogenic induced MSCs (Figure 2(a)). Furthermore, RT-PCR was performed to confirm the differentiation by assessing lineage specific gene expression, and amplified products of each gene in agarose gel were displayed in Figure 2(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.