Network of Excellence for Functional Biomaterials, National University of Ireland, NFB building, IDA business park, Dangan, Newcastle, Galway, Ireland.
Nanoscale. 2010 Dec;2(12):2718-23. doi: 10.1039/c0nr00456a. Epub 2010 Sep 8.
Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique hypothesized to allow quantification of both bound and unbound DNA. A comparison of the two methods was performed by quantifying the elution of six different varieties of DNA complexes from a model biomaterial (collagen) scaffold. After seven days of elution, the PicoGreen® assay only allowed detection of three types of complexes (those formed using Lipofectin™ and two synthesised copolymers). However, the Cy5 fluorescent labeling technique enabled detection of all six varieties including those formed via common transfection agents poly(ethylene imine), poly-L-lysine and SuperFect™. This allowed reliable quantification of the elution of all these complexes from the collagen scaffold. Thus, while intercalating dyes may be effective and reliable for detecting double-stranded, unbound DNA, the technique described in this work allowed reliable quantification of DNA independent of complexation state.
洗脱核酸的定量是表征基于生物材料的基因传递系统的关键参数。最常用的方法是使用嵌入荧光染料（如 PicoGreen®）检测样品。然而，该技术是为未结合的 DNA 开发的，而当前基因传递的趋势是用转染试剂使 DNA 浓缩，这会干扰嵌入。在这里，首次在复合物形成之前，用荧光染料 Cy5 对 DNA 进行永久性标记，这是一种假设可以定量结合和未结合 DNA 的替代技术。通过从模型生物材料（胶原）支架中定量洗脱六种不同类型的 DNA 复合物，比较了这两种方法。在洗脱 7 天后，PicoGreen®检测仅允许检测三种类型的复合物（使用 Lipofectin™和两种合成共聚物形成的复合物）。然而，Cy5 荧光标记技术能够检测所有六种类型的复合物，包括通过常用转染剂聚（亚乙基亚胺）、聚-L-赖氨酸和 SuperFect™形成的复合物。这使得能够从胶原支架中可靠地定量洗脱所有这些复合物。因此，虽然嵌入染料可能对检测双链、未结合的 DNA 有效且可靠，但本文所述的技术可以独立于复合物状态可靠地定量 DNA。
