Improved method to prepare RNA-free DNA from mammalian cells.
To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-3H]uridine-labeled nucleic acid with 50 microg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 microg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3' to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 microg/ml RNase T1, which preferentially cleaves RNA 3' to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-3H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.
Rubsam
L Z
LZ
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor 48109, USA.
Shewach
D S
DS
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004273:DNA, Neoplasm / Q000302:isolation & purification* / Q000378:metabolism; D005910:Glioma / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D051381:Rats; D015203:Reproducibility of Results; D006163:Ribonuclease T1 / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013936:Thymidine / Q000032:analysis / Q000378:metabolism; D014316:Tritium; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000032:analysis / Q000378:metabolism
D004273:DNA, Neoplasm; D014316:Tritium; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic; D013936:Thymidine; D014529:Uridine