Quantitation of digoxigenin-labeled DNA hybridized to DNA and RNA slot blots.
Quantitation of digoxigenin-labeled DNA hybridized to DNA and RNA slot blots.
复制标题
对与 DNA 和 RNA 狭缝印迹杂交的地高辛标记 DNA 进行定量。
DOI：
10.1006/abio.1995.1398
复制
发表时间：
1995
影响因子：
2.9
通讯作者：
Williams,DE
中科院分区：
文献类型：
--
作者：
Krueger,SK;Williams,DE
来自低丰度mrna的信息定量和有限的组织可用性需要灵敏的探针检测方法，准确的信号定量方法，以及剥离和重新探测膜的能力。利用兔含黄素单加氧酶基因家族的异构体FMO1 cDNA随机引物，标记地高高素探针，评价和优化了Genius系统对DNA和RNA缝隙印迹信号的定量分析。优化的标准是低信噪比，x射线胶片上条带的信号密度与核酸浓度线性增加，完全剥离膜，重新杂交后再现初始条带模式。用预杂交/杂交水溶液获得低信噪比。从带正电的膜上进行碱性剥离前后的DNA槽印迹成功定量。在剥离过程中，RNA槽印迹受到过度和不均匀的膜上RNA损失的影响。可靠的定量超过一个周期的检测需要高度带电的尼龙膜，并仔细剪裁RNA固定方法和碱性剥离条件。
Quantitation of message from low-abundance mRNAs and limited availability of tissues requires sensitive methods for probe detection, accurate methods for quantitation of signal, and the ability to strip and reprobe membranes. A random-primed, digoxigenin-labeled probe from cDNA of FMO1, an isoform of the flavin-containing monooxygenase gene family, from rabbit was used in the evaluation and optimization of the Genius system for quantitation of signal from DNA and RNA slot blots. Criteria for optimization were a low signal to noise ratio, a linear increase in density of signal vs nucleic acid concentration of bands on X-ray film, complete stripping of membranes, and reproduction of the initial banding pattern upon rehybridization. A low signal-to-noise ratio was obtained with an aqueous prehybridization/hybridization solution. DNA slot blots were successfully quantitated before and after alkaline stripping from positively charged membranes. RNA slot blots were subject to excessive and uneven loss of RNA from the membranes during stripping procedures. Reliable quantitation for more than one cycle of detection required highly charged nylon membranes, and careful tailoring of RNA fixation methods and alkaline stripping conditions.