Molecular Techniques and Methods

Removal of DNA from RNA Sample

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

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
  • This solution should be RNase-free.




    PROCEDURES

  • 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.

    2. Incubate the mixture for 15 minutes at 37oC.

    3. Extract the sample once with Acid phenol: chloroform: IAA (25: 24: 1).

    4. Extract the sample once with chloroform: IAA (24: 1).

    5. Add 1/10th volume of 3 M Sodium acetate (pH 7.0) and 2.5 volume of ethanol.

    6. Precipitate at -70oC for 1 hour.

    7. Centrifuge to pellet the RNA at 13,000g for 20 minutes at 4oC.

    8. Air dry the RNA pellet briefly.

    9. Resuspend the RNA in DEPC-treated H2O.

    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





  • KIT INFORMATION




    REFERENCES

  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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