Sensitivity of Prestaining RNA with Ethidium Bromide Before Electrophoresis and Performance of Subsequent Northern Blots Using Heterologous DNA Probes
Sensitivity of Prestaining RNA with Ethidium Bromide Before Electrophoresis and Performance of Subsequent Northern Blots Using Heterologous DNA Probes
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电泳前用溴化乙锭预染色 RNA 的灵敏度以及使用异源 DNA 探针进行后续 Northern 印迹的性能
DOI：
10.1007/s12033-012-9553-4
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发表时间：
2013-06-01
影响因子：
2.6
通讯作者：
Zhang, Nianhui
中科院分区：
文献类型：
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作者：
Zhao, Yun;Du, Linfang;Zhang, Nianhui
在运行甲醛琼脂糖凝胶之前，将低浓度的溴化乙酯（EtBr）添加到RNA样品中，可以在电泳过程中或电泳或将分离的RNA印迹到膜上后的任何时间检查RNA的完整性并评估大小分离的质量，而不会显着影响迁移性，转移或杂交。在本研究中，我们通过在电泳前加热含EtBr的RNA样品，在不同的变性条件下，系统地考察了影响RNA预染色敏感性的因素。我们还检测了etbr染色RNA与外源DNA探针的杂交效率。结果表明，EtBr染色RNA的荧光强度不仅受到先前报道的EtBr浓度的影响，还受到RNA数量、变性时间和变性温度的影响。事先用40 μ g/mL EtBr染色RNA显著降低了与异源DNA探针的Northern blot杂交效率。我们建议，为了最好地结合染色灵敏度和与异源DNA探针杂交的效率，用于保存RNA的EtBr浓度不应超过30 μ g/mL。etbr染色RNA的杂交效率不仅受到影响染色敏感性的因素的影响，还受到所用探针类型的影响。
Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde-agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 mu g/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to prestain RNA should not exceed 30 mu g/mL. The efficiency of the hybridization of EtBr-prestained RNA was affected not only by factors that affect staining sensitivity but also by the type of probe used.