Improved method to prepare RNA-free DNA from mammalian cells

Area of Science:

• Molecular Biology
• Biochemistry

Background:

• Ribonuclease A (RNase A) is commonly used to remove RNA from DNA during nucleic acid isolation.
• However, RNase A alone may not completely eliminate ribonucleotides, potentially affecting downstream analyses.
• High levels of residual ribonucleosides, particularly guanosine, have been observed after RNase A treatment.

Purpose of the Study:

• To evaluate the effectiveness of RNase A in removing RNA for DNA studies.
• To develop a more efficient method for RNA removal from nucleic acid extracts.
• To improve the detection of nucleoside analog incorporation into DNA.

Main Methods:

• Incubation of nucleic acid samples with varying concentrations of RNase A.
• Analysis of ribonucleoside levels using High-Performance Liquid Chromatography (HPLC).
• Addition of RNase T1 in combination with RNase A for RNA digestion.

Main Results:

• RNase A treatment alone resulted in significant residual ribonucleosides, with guanosine being a major component.
• Increasing RNase A concentration showed limited improvement in ribonucleoside removal.
• The combined use of RNase A and RNase T1 reduced total ribonucleosides by nearly 20-fold.
• A novel method achieved >99% RNA removal, with HPLC detecting contamination more sensitively than radiolabeling.

Conclusions:

• RNase A alone is insufficient for complete RNA removal in DNA preparations.
• A combination of RNase A and RNase T1 provides a rapid and highly effective method for RNA elimination.
• The developed method enhances the accuracy of nucleoside analog incorporation studies into DNA.