title: Validation of a PicoGreen-based DNA quantification integrated in an RNA extraction method for two-dimensional and three-dimensional cell cultures.
abstract: DNA measurement and RNA extraction are two frequently used methods for cell characterization. In the conventional protocols, they require similar, but separate samples and in most cases, different pretreatments. The few combined protocols that exist still include time-consuming steps. Hence, to establish an efficient combined RNA extraction and DNA measurement protocol for two-dimensional (2D) and three-dimensional (3D) cell cultures, a PicoGreen-based DNA measurement was integrated in an existing RNA extraction protocol. It was validated by analysis of the influence of different lysis buffers, RLT, RA1, or Trizol, used for RNA extraction on the measured DNA concentration. The DNA cell yield was evaluated both in cell suspensions (2D) and on 3D cell-seeded scaffolds. Results showed that the different RNA lysis buffers caused a concentration-dependent perturbation of the PicoGreen signal. The measured DNA concentrations in 2D and 3D using RLT and RA1 buffer were comparable, also to the positive control. We, therefore, concluded that RNA extraction protocols using RA1 or RLT buffer allow the integration of a DNA quantification step without the buffer influencing the results. Hence, the combined DNA measurement and RNA extraction offer an alternative for DNA measurement techniques that is time and sample saving, for both 2D cell cultures and specific 3D constructs.
authors.affiliation: Laboratory for Skeletal Development and Joint Disorders, KU Leuven, Leuven, Belgium.
authors.forename: Yantian
authors.initials: Y
authors.lastname: Chen
authors.forename: Maarten
authors.initials: M
authors.lastname: Sonnaert
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Roberts
authors.forename: Frank P
authors.initials: FP
authors.lastname: Luyten
authors.forename: Jan
authors.initials: J
authors.lastname: Schrooten
mesh_terms: D000497:Alloys; D002021:Buffers; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification*; D012015:Reference Standards; D054457:Tissue Scaffolds / Q000737:chemistry; D014025:Titanium / Q000737:chemistry
chemical_list: D000497:Alloys; D002021:Buffers; D009930:Organic Chemicals; C099476:PicoGreen; C031462:titanium alloy (TiAl6V4); D012313:RNA; D004247:DNA; D014025:Titanium