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

Abstract

Objectives: (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 andmRNA of normal cells and cancer cells, and (iii) to investigate whether there is some selectivity involved in the release of cfDNA. Design and 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 newcandidate 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