FLUOROMETRIC QUANTIFICATION OF DNA IN CELLS AND TISSUE
FLUOROMETRIC QUANTIFICATION OF DNA IN CELLS AND TISSUE
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DOI：
10.1016/0003-2697(83)90212-9
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发表时间：
1983-01-01
影响因子：
2.9
通讯作者：
WILFINGER, WW
中科院分区：
文献类型：
--
作者：
DOWNS, TR;WILFINGER, WW
描述了简单快速的 DNA 溶解程序的验证。通过在氢氧化铵-Triton X-100 溶液中裂解大鼠细胞或组织匀浆来定量提取完整的聚合 DNA。溶解过程可灭活内源性 DNA 酶并增加提取的 DNA 的荧光增强活性，从而无需酶处理或暴露于高盐溶液。提取物可直接用于双苯并咪唑 (Hoechst 33258) 试剂的灵敏荧光增强测定。 DNA 细胞含量的估计不受裂解细胞数量或测定中使用的裂解液体积的影响。在所有情况下，溶解的 DNA 估计值都是线性的，并且与牛 DNA 标准品平行。该测定中 DNA 估计的最佳范围是 5-150 ng。此外，用该方法获得的 DNA 估计值与标准二苯胺测定法非常一致。这种简单的一步 DNA 提取程序可与 Hoechst 试剂结合使用，以定量估计细胞或组织提取物中的 DNA 水平。
The validation of a simple and rapid DNA solubilization procedure is described. Quantitative extraction of intact, polymerized DNA was achieved by [rat] cell lysis or tissue homogenization in an ammonium hydroxide-Triton X-100 solution. The solubilization procedure inactivates endogenous DNase and increases the fluorescence-enhancement activity of the extracted DNA, thereby eliminating the need for enzyme treatment or exposure to high salt solutions. The extracts can be utilized directly in a sensitive fluorescence-enhancement assay with bisbenzimidazole (Hoechst 33258) reagent. Estimates of DNA cell content were unaffected by the number of cells lysed or the volume of lysate employed in the assay. In all cases, the solubilized DNA estimates were linear and parallel to the bovine DNA standard. The optimum range for estimation of DNA in this assay is 5-150 ng. In addition, estimates of DNA obtained with this method and the standard diphenylamine assay were in excellent agreement. This simple, 1-step DNA extraction procedure can be utilized in conjunction with Hoechst reagent to obtain quantitative estimates of DNA levels in cell or tissue extracts.