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Reference gene selection for in vitro cell-free DNA analysis and gene expression profiling.

Abel Jacobus Bronkhorst1, Janine Aucamp1, Johannes F Wentzel2

  • 1Centre for Human Metabolomics, Biochemistry Division, North-West University, Potchefstroom 2520, South Africa.

Clinical Biochemistry
|February 7, 2016
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Summary

Beta-actin (ACTB) is an optimal housekeeping gene (HKG) for cell-free DNA (cfDNA) and mRNA quantification. Results indicate cfDNA is selectively released from cells, offering insights for gene expression profiling.

Keywords:
BiomarkersCancerCell-free DNAHousekeeping genesReal-time PCRTumor markers

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Area of Science:

  • Molecular Biology
  • Genomics
  • Cancer Research

Background:

  • Accurate quantification of cell-free DNA (cfDNA) and mRNA is crucial for gene expression profiling and cancer diagnostics.
  • Selection of appropriate housekeeping genes (HKGs) is essential for reliable normalization in quantitative PCR (qPCR).
  • Understanding the release mechanisms of cfDNA can provide insights into cellular processes and disease states.

Purpose of the Study:

  • To optimize quantification of cfDNA and mRNA using eight HKGs.
  • To compare HKG occurrence in cfDNA and mRNA from normal and cancer cells.
  • To investigate selective release of cfDNA from cells.

Main Methods:

  • cfDNA isolation from cell culture medium of cancer and non-malignant cell lines.
  • mRNA isolation and cDNA synthesis from the same cells.
  • Real-time PCR amplification of cfDNA and cDNA using eight different HKGs.

Main Results:

  • Beta-actin (ACTB) emerged as the most suitable HKG for cfDNA and mRNA quantification across all tested cell lines.
  • No significant difference in HKG occurrence was observed between cancer and healthy cells.
  • A consistent and distinct difference was found between mRNA expression and cfDNA profiles for all cell lines.

Conclusions:

  • ACTB is a strong candidate HKG for robust cfDNA analysis and gene expression profiling.
  • The findings suggest selective release of cfDNA from cells into the culture medium.
  • This study provides a validated HKG for improved accuracy in molecular diagnostics.