Related Experiment Videos
Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase I digestion
P Bauer1, A Rolfs, V Regitz-Zagrosek
1Humboldt Universität, Berlin, Germany.
Abstract:
The precise quantification of rare mRNA copies from intronless genes by reverse transcription polymerase chain reaction (RT-PCR) requires the complete removal of genomic DNA because discrimination of cDNA and DNA amplification products by differing sizes of PCR products is not possible. Elimination of DNA is achieved by treating the RNA sample with RNase-free DNase I before RT-PCR. The lack of a PCR product from DNase-treated RNA samples before RT is usually accepted as a proof of efficient DNA destruction. However, this may vary depending on the metal cofactor used in the DNase I cleavage. Treating DNA-contaminated RNA samples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after additional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor, RT-created DNA fragments do not appear. This is because in the presence of manganese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended or that have protruding termini of only one or two nucleotides in length. However, overlapping fragments with the potential to recombine are created by DNase digestion with magnesium as cofactor. Because one cannot differentiate between a PCR signal produced by RNA and one produced by recombined DNA after DNase I digestion and RT, all such DNase I assays should be performed with manganese instead of magnesium.
Insights
To accurately quantify rare mRNA using RT-PCR, complete genomic DNA removal is crucial. Using manganese as a cofactor during DNase I treatment ensures efficient DNA elimination, preventing artifactual amplification and ensuring reliable results.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Accurate quantification of rare mRNA via RT-PCR necessitates complete removal of contaminating genomic DNA.
- Genomic DNA contamination can lead to false-positive results in RT-PCR, especially for intronless genes where cDNA and DNA products are indistinguishable by size.
Purpose of the Study:
- To investigate the impact of different metal cofactors (magnesium vs. manganese) on DNase I digestion efficiency for genomic DNA removal prior to RT-PCR.
- To determine the optimal conditions for DNase I treatment to prevent artifactual DNA amplification and ensure accurate mRNA quantification.
Main Methods:
- RNA samples contaminated with genomic DNA were treated with RNase-free DNase I using either magnesium or manganese as a cofactor.
- Treated samples underwent PCR without reverse transcription (RT) to assess DNA elimination.
- Samples were subsequently subjected to RT-PCR to evaluate the impact of DNase I treatment on RNA amplification and potential DNA re-amplification.
Main Results:
- DNase I treatment with magnesium as a cofactor resulted in DNA fragments that could be re-amplified during subsequent RT-PCR, leading to inaccurate quantification.
- DNase I treatment with manganese as a cofactor effectively cleaved DNA into blunt-ended fragments, preventing re-amplification and ensuring accurate RNA detection.
- The choice of cofactor significantly influences the integrity of DNA after DNase I digestion and its potential for artifactual amplification.
Conclusions:
- Manganese is the preferred cofactor for DNase I treatment when aiming for complete genomic DNA elimination prior to RT-PCR.
- Using manganese prevents the formation of overlapping DNA fragments that can recombine and lead to false-positive signals, thereby ensuring the reliability of rare mRNA quantification.
- All DNase I assays for genomic DNA removal in RT-PCR protocols should be performed with manganese instead of magnesium.