A "Referral from the Doctor" Blog Article-
Genomic DNA contamination has been a hot topic for qPCR researchers since the beginning. It is impossible to remove all DNA from RNA preparations without additional steps. The most commonly used technique is to incubate with DNAse I. The deoxyribonuclease I (DNAse I) enzyme catalyzes the hydrolytic cleavage of phosphodiester linkages adjacent to a pyrimidine nucleotide. It acts on single-stranded DNA, double-stranded DNA, and chromatin. DNAse I activity will degrade trace amounts of genomic DNA (up to 10 µg/mL) that could otherwise result in falsely positive signals in subsequent RT-PCR reactions. DNAse I is a heat-inactivated nuclease, requiring both the presence of EDTA and temperatures of 75oC for 5 minutes for complete inactivation. The extreme temperatures associated with heat-inactivation of the enzyme may cause damage to the RNA through chemical mediated degradation if even small amounts of metal ions are present. Lower temperatures will not fully inactivate the DNAse before reverse transcription of the RNA to cDNA. Because we cannot discriminate between cDNA and DNA amplification products, initial copy number determination is compromised.
While acid phenol extraction has been a reliable alternative method for DNA removal from nucleic acid preparations, the formation of an organic phase and the resultant hazards associated with working with organic wastes, has made this technique obsolete. Other techniques such as using immobilized DNAses or the use of manganese instead of magnesium in the DNAse I incubation have contributed significantly to solving the issue of genomic DNA carryover contamination.
The little known alternative is to reduce contaminating genomic DNA with a Lithium Chloride (LiCl) precipitation (ppt). LiCl ppt can be used after DNAse I treatment to inactivate the enzyme without using heat-inactivation. LiCl ppt will selectively precipitate full-length RNA transcripts from solution to the exclusion of protein or DNA and is effective at removing free nucleotides. The LiCl ppt method requires only low concentrations, 2.5M, of LiCl and short, 30 minute, room temperature incubations. RNA recovery from the precipitant solution is dependent upon centrifugation conditions. Centrifugation at high speeds around 16K x g, for 20 minutes at 40C will enable the recovery of as little as 50ng of RNA. For an inexpensive, reproducible, efficient way to purify your RNA after isolation and treatment with DNAse I, try the Lithium Chloride precipitation method.
Written by: Christina Ferrell, Ph.D., Technical Applications Specialist
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