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Simultaneous detection of mRNA and protein stem cell markers in live cells.

Rhee WJ, Bao G - BMC Biotechnol. (2009)

Bottom Line: We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells.Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol.We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA.

ABSTRACT

Background: Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation.

Results: Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology.

Conclusion: Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS) cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA) and cell-surface (protein) stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

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Related in: MedlinePlus

The cDNA sequence of mouse Oct-4 mRNA and the hybridization sites of Oct-4 targeting molecular beacons (MB1 – MB13).
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Figure 2: The cDNA sequence of mouse Oct-4 mRNA and the hybridization sites of Oct-4 targeting molecular beacons (MB1 – MB13).

Mentions: To ensure that there is good target accessibility in live-cell detection of Oct-4 mRNA, we have designed and tested 13 molecular beacons (designated as MB1 to MB13) that target different sites on Oct-4 mRNA, as shown in Figure 2. Target accessibility is one of the most important issues in live-cell mRNA detection since, although a molecular beacon can be designed to have its probe sequence unique to the target mRNA, the target sequence may not necessarily be accessible in a living cell. Specifically, the probes need to avoid targeting sequences that form secondary structures or are occupied by RNA-binding proteins [26] in order to have a good level off signal.


Simultaneous detection of mRNA and protein stem cell markers in live cells.

Rhee WJ, Bao G - BMC Biotechnol. (2009)

The cDNA sequence of mouse Oct-4 mRNA and the hybridization sites of Oct-4 targeting molecular beacons (MB1 – MB13).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2682800&req=5

Figure 2: The cDNA sequence of mouse Oct-4 mRNA and the hybridization sites of Oct-4 targeting molecular beacons (MB1 – MB13).
Mentions: To ensure that there is good target accessibility in live-cell detection of Oct-4 mRNA, we have designed and tested 13 molecular beacons (designated as MB1 to MB13) that target different sites on Oct-4 mRNA, as shown in Figure 2. Target accessibility is one of the most important issues in live-cell mRNA detection since, although a molecular beacon can be designed to have its probe sequence unique to the target mRNA, the target sequence may not necessarily be accessible in a living cell. Specifically, the probes need to avoid targeting sequences that form secondary structures or are occupied by RNA-binding proteins [26] in order to have a good level off signal.

Bottom Line: We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells.Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol.We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA.

ABSTRACT

Background: Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation.

Results: Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology.

Conclusion: Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS) cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA) and cell-surface (protein) stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

Show MeSH
Related in: MedlinePlus