Comparison of Milli-Q PF plus water with DEPC-treated water in the preparation and analysis of RNA.
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 (HCCSMC). RNA was prepared by solubilization in guanidinium isothiocyanate, phenol/chloroform extraction, and isopropanol precipitation followed by Northern blot analysis. Hybridization with fibronectin (approximately 7.6kb) and glyceraldehyde-3-phosphate dehydrogenase (1.2kb) 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 or 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 or Milli-Q PF water. We conclude that Milli-Q PF water is an acceptable substitute to DEPC-treated water for the preparation of RNA and Northern blot analysis.
Huang
Y H
YH
Department of Urology, Boston University Medical Center, MA 02118, USA.
Leblanc
P
P
Apostolou
V
V
Stewart
B
B
Moreland
R B
RB
D000818:Animals; D004047:Diethyl Pyrocarbonate; D004355:Drug Stability; D004791:Enzyme Inhibitors; D005069:Evaluation Studies as Topic; D006801:Humans; D008722:Methods; D008851:Micropore Filters; D012313:RNA / Q000737:chemistry* / Q000302:isolation & purification*; D011817:Rabbits; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D014867:Water / Q000737:chemistry*
D004791:Enzyme Inhibitors; D014867:Water; D012313:RNA; D012260:Ribonucleases; D004047:Diethyl Pyrocarbonate