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Updated: Jun 12, 2026

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR
09:03

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Published on: May 29, 2014

A quantitative method to evaluate mesenchymal stem cell lipofection using real-time PCR.

S C Ribeiro1, R Mendes, C Madeira

  • 1IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.

Biotechnology Progress
|June 10, 2010
PubMed
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This study introduces a new quantitative real-time PCR (RT-PCR) method to measure plasmid DNA in human mesenchymal stem cells (MSCs). This technique accurately quantifies DNA uptake, improving gene delivery strategies for stem cell therapy.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Stem Cell Research

Background:

  • Genetic modification of human mesenchymal stem cells (MSCs) enhances therapeutic potential and understanding of cell regulation.
  • Efficient nonviral gene delivery systems are crucial for MSC modification.
  • Current methods often rely on reporter gene expression, not direct DNA uptake quantification.

Purpose of the Study:

  • To establish a quantitative real-time PCR (RT-PCR) method for determining intracellular plasmid DNA copy number in human MSCs.
  • To evaluate the accuracy and efficiency of this new method for assessing gene delivery.
  • To compare RT-PCR results with traditional flow cytometry methods.

Main Methods:

  • Developed a novel RT-PCR assay for quantifying intracellular plasmid DNA in human MSCs post-lipofection.

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An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios
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Published on: December 8, 2023

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Last Updated: Jun 12, 2026

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR
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Published on: May 29, 2014

An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios
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  • The method bypasses the need for cell lysis or DNA purification.
  • Assessed the influence of cell number on RT-PCR sensitivity and validated its linear range (75–2.5 x 10⁶ copies/cell).

    • Main Results:

    • The RT-PCR method demonstrated high reproducibility, sensitivity, and a broad linear range.
    • Compared to flow cytometry, RT-PCR provided a more accurate measure of plasmid cellular uptake.
    • Flow cytometry results did not always correlate directly with the actual amount of plasmid DNA internalized by MSCs.

    Conclusions:

    • Established a rapid and quantitative assay for intracellular plasmid DNA in stem cells.
    • This method is highly beneficial for optimizing gene delivery strategies in MSCs.
    • The findings highlight the limitations of reporter gene assays and emphasize the importance of direct DNA quantification.