title: Reference gene selection for in vitro cell-free DNA analysis and gene expression profiling.
abstract: OBJECTIVE
  (i) To optimize cell-free DNA (cfDNA) and mRNA quantification using eight housekeeping genes (HKGs), (ii) to determine if there is a difference in the occurrence of HKGs in the cfDNA and mRNA of normal cells and cancer cells, and (iii) to investigate whether there is some selectivity involved in the release of cfDNA.
  
  
  METHODS
  cfDNA was isolated directly from the growth medium of 3 cultured cancer cell lines and one non-malignant, primary cell line. At the same time interval, mRNA was isolated from these cells and cDNA was synthesized. CfDNA and cDNA were then amplified with real-time PCR utilizing eight different HKGs.
  
  
  RESULTS
  For all cell lines tested, Beta-actin (ACTB) is the most appropriate HKG to use as a control for cfDNA and mRNA quantification. There was no clear difference in the occurrence of HKGs between cancer cells and healthy cells. Lastly, there is a consistent and distinct difference between the mRNA expression and cfDNA of all cell lines.
  
  
  CONCLUSIONS
  This study reveals a new candidate HKG for a robust control in cfDNA analysis and gene expression profiling, and should be considered for optimal analysis. Furthermore, results indicate that cfDNA is selectively released from cells into culture medium.
authors: Centre for Human Metabolomics, Biochemistry Division, North-West University, Potchefstroom 2520, South Africa. Electronic address: abel.bronkhorst29@gmail.com.
authors: Abel Jacobus
authors: AJ
authors: Bronkhorst
authors: Centre for Human Metabolomics, Biochemistry Division, North-West University, Potchefstroom 2520, South Africa.
authors: Janine
authors: J
authors: Aucamp
authors: Centre of Excellence for Pharmaceutical Sciences (PHARMACEN), North-West University, Potchefstroom 2520, South Africa.
authors: Johannes F
authors: JF
authors: Wentzel
authors: Centre for Human Metabolomics, Biochemistry Division, North-West University, Potchefstroom 2520, South Africa.
authors: Piet J
authors: PJ
authors: Pretorius
mesh_terms: D015415:Biomarkers / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis* / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling; D020043:Genes, Essential / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012867:Skin / Q000166:cytology / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger; D004247:DNA
keywords: Biomarkers; Cancer; Cell-free DNA; Housekeeping genes; Real-time PCR; Tumor markers