M Rohan Fernando1, Chao Jiang2, Gary D Krzyzanowski3, Wayne L Ryan4. 1. Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE, USA; Department of Research and Development, CFGenome, Omaha, NE, USA. Electronic address: mrohan.fernando@unmc.edu. 2. Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE, USA. 3. Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, NE, USA; Department of Research and Development, CFGenome, Omaha, NE, USA. 4. Department of Research and Development, CFGenome, Omaha, NE, USA.
Abstract
BACKGROUND: Plasma cell-free DNA (cfDNA) fragment size distribution provides important information required for diagnostic assay development. We have developed and optimized droplet digital PCR (ddPCR) assays that quantify short and long DNA fragments. These assays were used to analyze plasma cfDNA fragment size distribution in human blood. METHODS: Assays were designed to amplify 76,135, 490 and 905 base pair fragments of human β-actin gene. These assays were used for fragment size analysis of plasma cell-free, exosome and apoptotic body DNA obtained from normal and pregnant donors. RESULTS: The relative percentages for 76, 135, 490 and 905 bp fragments from non-pregnant plasma and exosome DNA were 100%, 39%, 18%, 5.6% and 100%, 40%, 18%,3.3%, respectively. The relative percentages for pregnant plasma and exosome DNA were 100%, 34%, 14%, 23%, and 100%, 30%, 12%, 18%, respectively. The relative percentages for non-pregnant plasma pellet (obtained after 2nd centrifugation step) were 100%, 100%, 87% and 83%, respectively. CONCLUSION: Non-pregnant Plasma cell-free and exosome DNA share a unique fragment distribution pattern which is different from pregnant donor plasma and exosome DNA fragment distribution indicating the effect of physiological status on cfDNA fragment size distribution. Fragment distribution pattern for plasma pellet that includes apoptotic bodies and nuclear DNA was greatly different from plasma cell-free and exosome DNA.
BACKGROUND: Plasma cell-free DNA (cfDNA) fragment size distribution provides important information required for diagnostic assay development. We have developed and optimized droplet digital PCR (ddPCR) assays that quantify short and long DNA fragments. These assays were used to analyze plasma cfDNA fragment size distribution in human blood. METHODS: Assays were designed to amplify 76,135, 490 and 905 base pair fragments of human β-actin gene. These assays were used for fragment size analysis of plasma cell-free, exosome and apoptotic body DNA obtained from normal and pregnant donors. RESULTS: The relative percentages for 76, 135, 490 and 905 bp fragments from non-pregnant plasma and exosome DNA were 100%, 39%, 18%, 5.6% and 100%, 40%, 18%,3.3%, respectively. The relative percentages for pregnant plasma and exosome DNA were 100%, 34%, 14%, 23%, and 100%, 30%, 12%, 18%, respectively. The relative percentages for non-pregnant plasma pellet (obtained after 2nd centrifugation step) were 100%, 100%, 87% and 83%, respectively. CONCLUSION: Non-pregnant Plasma cell-free and exosome DNA share a unique fragment distribution pattern which is different from pregnant donor plasma and exosome DNA fragment distribution indicating the effect of physiological status on cfDNA fragment size distribution. Fragment distribution pattern for plasma pellet that includes apoptotic bodies and nuclear DNA was greatly different from plasma cell-free and exosome DNA.
Authors: Boglárka Vincze; András Gáspárdy; Alexandra Biácsi; Endre Ákos Papp; László Garamvölgyi; Endre Sós; Sándor Cseh; Gábor Kovács; Zsolt Pádár; Petra Zenke Journal: Sci Rep Date: 2019-10-24 Impact factor: 4.379
Authors: Vida Ungerer; Abel J Bronkhorst; Priscilla Van den Ackerveken; Marielle Herzog; Stefan Holdenrieder Journal: Sci Rep Date: 2021-05-04 Impact factor: 4.379