Evaluation of normalization strategies for qPCR quantitation of intracellular viral DNA: The example of Vaccinia virus
ABSTRACT Quantitation of intracellular viral genomes is critical in both clinical and fundamental virology. Quantitative real time PCR (qPCR) is currently the gold standard to detect and monitor virus infections, due to its high sensitivity and reproducibility. The reliability of qPCR data depends primarily on the technical process. Normalization, which corrects inter-sample variations related to both pre-analytical and qPCR steps, is a key point of an accurate quantitation. Total DNA input and qPCR-measured standards were evaluated to normalize intracellular Vaccinia virus (VACV) genomes. Three qPCR assays targeting either a single-copy chromosomic gene, a repeated chromosomic DNA sequence, or a mitochondrial DNA sequence were compared. qPCR-measured standards, unlike total DNA input, allowed for accurate normalization of VACV genome, regardless of the cell number. Among PCR-measured standards, chromosomic DNA and mitochondrial DNA were equivalent to normalize VACV DNA and multi-copy standards displayed lower limits of quantitation than single-copy standards. The combination of two qPCR-measured standards slightly improved the reliability of the normalization. Using one or two multi-copy standards must be favored for relative quantitation of intracellular VACV DNA. This concept could be applied to other DNA viruses.
- SourceAvailable from: Tzachi Bar[Show abstract] [Hide abstract]
ABSTRACT: Real-time PCR is becoming the method of choice for precise quantification of minute amounts of nucleic acids. For proper comparison of samples, almost all quantification methods assume similar PCR efficiencies in the exponential phase of the reaction. However, inhibition of PCR is common when working with biological samples and may invalidate the assumed similarity of PCR efficiencies. Here we present a statistical method, Kinetic Outlier Detection (KOD), to detect samples with dissimilar efficiencies. KOD is based on a comparison of PCR efficiency, estimated from the amplification curve of a test sample, with the mean PCR efficiency of samples in a training set. KOD is demonstrated and validated on samples with the same initial number of template molecules, where PCR is inhibited to various degrees by elevated concentrations of dNTP; and in detection of cDNA samples with an aberrant ratio of two genes. Translating the dissimilarity in efficiency to quantity, KOD identifies outliers that differ by 1.3-1.9-fold in their quantity from normal samples with a P-value of 0.05. This precision is higher than the minimal 2-fold difference in number of DNA molecules that real-time PCR usually aims to detect. Thus, KOD may be a useful tool for outlier detection in real-time PCR.Nucleic Acids Research 10/2003; 31(17):e105. · 9.11 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Quantitative analysis of mitochondrial DNA (mtDNA) and its common deletion (CD) are sensitive and early markers for mitochondrial mutations and suffering. However, the use of purified DNA can lead to quantification errors because of variable DNA extraction yields due to the significant differences in size and structure between genomic DNA (gDNA) and mtDNA. We report a real-time qPCR-based protocol directly on tissue lysate, without DNA extraction. This method, which allows both absolute and relative measure, increases the measuring accuracy of the mtDNA/gDNA ratio and leads to reliable and more reproducible results when measuring the deleted/total mtDNA ratio.Analytical Biochemistry 10/2010; 409(2):298-300. DOI:10.1016/j.ab.2010.10.010 · 2.31 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Fibroblasts infected by Human Cytomegalovirus (CMV) undergo a robust increase in mitochondrial biogenesis with a corresponding increase in mitochondrial activity that is partly dependent on the viral anti-apoptotic pUL37x1 protein (vMIA). The increased respiration activity is blocked by the mitochondrial translation inhibitor chloramphenicol, which additionally suppresses viral production. Intriguingly, chloramphenicol and pUL37x1 depletion have different effects on respiration capacity but similar effects on CMV production, suggesting that pUL37x1 promotes viral replication by efficient utilization of new mitochondria. These results argue for a role of pUL37x1 beyond controlling apoptosis.Mitochondrion 09/2011; 11(6):935-45. DOI:10.1016/j.mito.2011.08.008 · 3.52 Impact Factor