Improved method to prepare RNA-free DNA from mammalian cells

Journal of Chromatography. B, Biomedical Sciences and Applications
L Z Rubsam, D S Shewach

Abstract

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 labelin...Continue Reading

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Citations

Dec 8, 2000·Biochemical Pharmacology·W B ParkerJ A Secrist
Nov 18, 2005·Plant Physiology and Biochemistry : PPB·Jason W JohnstonErica E Benson
Jun 10, 2008·Plant Physiology and Biochemistry : PPB·Rodrigo HasbúnRoberto Rodríguez
Aug 15, 2009·Lab on a Chip·Carol W PriceJames P Landers

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