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Selective silencing of DNA topoisomerase IIβ in human mesenchymal stem cells by siRNAs (small interfering RNAs).

Kamaci N, Emnacar T, Karakas N, Arikan G, Tsutsui K, Isik S - Cell Biol Int Rep (2010) (2011)

Bottom Line: A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos.Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs.The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Fatih University, Stem Cell Research Laboratory, 34500 Buyukcekmece, Istanbul, Turkey.

ABSTRACT
hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIβ is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIβ selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

No MeSH data available.


Related in: MedlinePlus

Cytotoxicity and cell viability assays of hMSCs after siRNA transfections with Lipofectamine RNAiMAXCytotoxicity of transfected hMSCs increases in the first 24 h in a dose-dependent manner and then become stabilized in a time-dependent manner (A). Cell viability assay of siRNA transfected hMSCs with Lipofectamine RNAiMAX reagent. Cell viability was affected by Lipofectamine RNAiMAX 20–30% in hMSCs (B). Transfections were performed twice at 0 and 48 h.
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Figure 4: Cytotoxicity and cell viability assays of hMSCs after siRNA transfections with Lipofectamine RNAiMAXCytotoxicity of transfected hMSCs increases in the first 24 h in a dose-dependent manner and then become stabilized in a time-dependent manner (A). Cell viability assay of siRNA transfected hMSCs with Lipofectamine RNAiMAX reagent. Cell viability was affected by Lipofectamine RNAiMAX 20–30% in hMSCs (B). Transfections were performed twice at 0 and 48 h.

Mentions: The LDH cytotoxicity detection kit is a colorimetric assay for the quantification of cell death and cell lysis, based on the determination of LDH activity released from the cytosol of damaged cells into the medium, thus indicating cell membrane damage. The LDH cytotoxicity assay kit was used following the manufacturer's instructions. The integrity of the plasma membrane was evaluated by an LDH assay following 12, 24, 36, 48, 60 and 72 h incubating the cells with 8, 25 and 50 nM of topo IIβ-specific siRNAs by Lipofectamine RNAiMAX transfection reagent (Figure 4A). Relative cytotoxicity of the reagent in transfected hMSCs increases in the first 24 h in a dose-dependent manner and then become stabilized in a time-dependent manner. The percentage of the damaged cells by the reagent during this time period was approximately in the range of 3.5–4.5%. These results demonstrated that Lipofectamine RNAiMAX did not cause considerable damage to the plasma membrane of hMSCs. The proliferation assay was performed to elucidate the effect of the same transfection reagent on hMSCs. We established a similar experimental setup as we did for the LDH assay to compare the proliferation rates of the cells treated with 8, 25 and 50 nM of siRNAs after 24, 48 and 72 h of transfection. A graph of the transfection reagent-treated and untreated samples revealed that cell viability of transfected cells decreased about 20% compared with control cells with 8 nM siRNA concentration (Figure 4B). The increased concentration of topo IIβ-specific siRNAs (25 and 50 nM) did not cause a significant difference in the viability of the transfected cells compared with the lower concentration.


Selective silencing of DNA topoisomerase IIβ in human mesenchymal stem cells by siRNAs (small interfering RNAs).

Kamaci N, Emnacar T, Karakas N, Arikan G, Tsutsui K, Isik S - Cell Biol Int Rep (2010) (2011)

Cytotoxicity and cell viability assays of hMSCs after siRNA transfections with Lipofectamine RNAiMAXCytotoxicity of transfected hMSCs increases in the first 24 h in a dose-dependent manner and then become stabilized in a time-dependent manner (A). Cell viability assay of siRNA transfected hMSCs with Lipofectamine RNAiMAX reagent. Cell viability was affected by Lipofectamine RNAiMAX 20–30% in hMSCs (B). Transfections were performed twice at 0 and 48 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475440&req=5

Figure 4: Cytotoxicity and cell viability assays of hMSCs after siRNA transfections with Lipofectamine RNAiMAXCytotoxicity of transfected hMSCs increases in the first 24 h in a dose-dependent manner and then become stabilized in a time-dependent manner (A). Cell viability assay of siRNA transfected hMSCs with Lipofectamine RNAiMAX reagent. Cell viability was affected by Lipofectamine RNAiMAX 20–30% in hMSCs (B). Transfections were performed twice at 0 and 48 h.
Mentions: The LDH cytotoxicity detection kit is a colorimetric assay for the quantification of cell death and cell lysis, based on the determination of LDH activity released from the cytosol of damaged cells into the medium, thus indicating cell membrane damage. The LDH cytotoxicity assay kit was used following the manufacturer's instructions. The integrity of the plasma membrane was evaluated by an LDH assay following 12, 24, 36, 48, 60 and 72 h incubating the cells with 8, 25 and 50 nM of topo IIβ-specific siRNAs by Lipofectamine RNAiMAX transfection reagent (Figure 4A). Relative cytotoxicity of the reagent in transfected hMSCs increases in the first 24 h in a dose-dependent manner and then become stabilized in a time-dependent manner. The percentage of the damaged cells by the reagent during this time period was approximately in the range of 3.5–4.5%. These results demonstrated that Lipofectamine RNAiMAX did not cause considerable damage to the plasma membrane of hMSCs. The proliferation assay was performed to elucidate the effect of the same transfection reagent on hMSCs. We established a similar experimental setup as we did for the LDH assay to compare the proliferation rates of the cells treated with 8, 25 and 50 nM of siRNAs after 24, 48 and 72 h of transfection. A graph of the transfection reagent-treated and untreated samples revealed that cell viability of transfected cells decreased about 20% compared with control cells with 8 nM siRNA concentration (Figure 4B). The increased concentration of topo IIβ-specific siRNAs (25 and 50 nM) did not cause a significant difference in the viability of the transfected cells compared with the lower concentration.

Bottom Line: A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos.Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs.The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Fatih University, Stem Cell Research Laboratory, 34500 Buyukcekmece, Istanbul, Turkey.

ABSTRACT
hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIβ is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIβ selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

No MeSH data available.


Related in: MedlinePlus