It is necessary to remove DNA contamination from RNA samples for
any of several reasons, especially when preparing for RNA-based
polymerase chain reaction (RT-PCR). This presents a significant compromise to the quantitative
nature of RNA-based assays; probes and primers often have great
difficulty distinguishing between RNA targets and DNA targets,
when both are present. A brief incubation of the sample(s) with
ribonuclease-free deoxyribonuclease I (RNase-free DNase I) will eliminate DNA by nuclease digestion. Although the resulting
RNA solution can be used directly, it should be extracted with
phenol: chloroform: IAA, and concentrated by salt and ethanol
precipitation.
Commercial preparations of RNase-free DNase I are purified to remove all traces of detectable RNase, and are
certified by the manufacturer to harbor no intrinsic RNase activity.
These preparations are very handy in the laboratory, and well
worth the purchase cost. There is no easy, cost-effective way
to prepare RNase-free DNase in the laboratory because of the labile
nature of DNase I.
MATERIALS AND SOLUTIONS
10 X DNase I Buffer (1 ml)
400 mM Tris-HCl (pH 7.9) -------------------- 400 ul of 1 M Tris-HCl
100 mM NaCl --------------------------------- 20 ul of 5 M NaCl
100 mM CaCl2 -------------------------------- 100 ul of 1 M CaCl2
50 mM MgCl2 --------------------------------- 50 ul of 1 M MgCl2 DEPC-treated H2O ---------------------------- 430 ul
All manipulations should be performed on ice! Always wear gloves!
1. To a sample of RNA suspected to be contaminated with DNA, add
10 X DNase I buffer and RNase-free DNase I to a final concentration
of about 3 U/ ug nucleic acid.
10. Assess the extent of DNA digestion as well as the integrity
of the RNA in the sample by electrophoresing an aliquot of the sample under denaturing conditions.
NOTES
The 1 unit of RNase-free DNase I will degrade 1 ug of DNA in 10 minutes at 37oC, performed in a 20-50 ul reaction volume.
DNase I is strongly inhibited in buffers containing 25 mM EDTA,
sodium dodecyl sulfate, and other denaturants, or if heated above
37oC