Huang Y H, Leblanc P, Apostolou V, Stewart B, Moreland R B
Boston University Medical Center, MA, USA.
Biotechniques. 1995 Oct;19(4):656-61.
The Milli-Q PF Plus water-polishing system is equipped with high-purity ion and organic removal media and a capillary fiber ultrafiltration device. The system produces ultrapure water practically free of ribonuclease contamination. The necessity for diethyl pyrocarbonate (DEPC)-treated solutions in RNA molecular biological procedures was tested by preparing RNA from a variety of tissues and tissue-cultured cells using either DEPC-treated, autoclaved solutions or pure Milli-Q PF water dispensed directly from the system. Tissue sources included rabbit brain, heart, lung, liver kidney and bladder as well as cultured human corpus cavernosum smooth muscle cells. RNA was prepared by guanidinium isothiocyanate solubilization, phenol/chloroform extraction and isopropanol precipitation followed by Northern blot analysis. Hybridization with fibronectin (ca. 7.6 kb) and glyceraldehyde-3-phosphate dehydrogenase (1.2 kb) revealed that water from a Milli-Q PF water system performed as well as DEPC-treated, autoclaved solutions. RNA stability at 37 degrees C was examined for various times using rabbit lung RNA in either DEPC-treated water for Milli-Q PF water. Intact RNA was detected after 6 hours in total RNA and by Northern blots hybridized with fibronectin. There was no significant difference in RNA degradation between DEPC-treated water and Milli-Q PF water. We conclude that Milli-Q PF water is an acceptable substitute for DEPC-treated water for the preparation of RNA and Northern blot analysis.
密理博PF Plus水净化系统配备了高纯度离子和有机污染物去除介质以及毛细管纤维超滤装置。该系统可生产几乎不含核糖核酸酶污染的超纯水。通过使用经焦碳酸二乙酯（DEPC）处理、高压灭菌的溶液或直接从该系统中取出的纯密理博PF水，从多种组织和组织培养细胞中制备RNA，测试了在RNA分子生物学实验中使用DEPC处理溶液的必要性。组织来源包括兔脑、心脏、肺、肝脏、肾脏和膀胱以及培养的人阴茎海绵体平滑肌细胞。通过异硫氰酸胍溶解、苯酚/氯仿提取和异丙醇沉淀制备RNA，随后进行Northern印迹分析。与纤连蛋白（约7.6 kb）和甘油醛-3-磷酸脱氢酶（1.2 kb）杂交显示，密理博PF水系统的水与经DEPC处理、高压灭菌的溶液效果相同。使用兔肺RNA在经DEPC处理的水中或密理博PF水中，在37℃下对RNA稳定性进行了不同时间的检测。在总RNA中6小时后以及与纤连蛋白杂交的Northern印迹中均检测到完整的RNA。经DEPC处理的水和密理博PF水之间在RNA降解方面没有显著差异。我们得出结论，密理博PF水是用于制备RNA和Northern印迹分析的DEPC处理水的可接受替代品。
