Abstract:We compared different methods (absorbance, fluorescent dye-binding, and digital PCR) for measuring the concentrations of human genomic DNA from cultured cells and absorbance measurements of a synthetic DNA oligonucleotide. NIST Standard Reference Material (SRM) 2082, a pathlength absorbance standard, was used to benchmark the absorbance measurements done with microvolume spectrophotometers and a microvolume plate reader. Control absorbance values were measured on a high accuracy spectrophotometer and a NIST ca… Show more
“… 27 Even with pristine genomic DNA extracted from cells, in our experience, the quantitation of genomic DNA can be a challenge. 31 Absorbance measurements are straightforward but require pure DNA of sufficient concentration to produce good results. Fluorescence dye binding methods widely used are sensitive but can be biased by contaminants or degraded DNA.…”
Section: Discussionmentioning
confidence: 99%
“…The dPCR assays used an input of 20 ng of DNA, using the mass of a normal human haploid genome of 3.3 pg, 31 which is equivalent to approximately 6060 genomes. The 0.125% VAF sample would be expected to have approximately seven variant copies.…”
We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.
“… 27 Even with pristine genomic DNA extracted from cells, in our experience, the quantitation of genomic DNA can be a challenge. 31 Absorbance measurements are straightforward but require pure DNA of sufficient concentration to produce good results. Fluorescence dye binding methods widely used are sensitive but can be biased by contaminants or degraded DNA.…”
Section: Discussionmentioning
confidence: 99%
“…The dPCR assays used an input of 20 ng of DNA, using the mass of a normal human haploid genome of 3.3 pg, 31 which is equivalent to approximately 6060 genomes. The 0.125% VAF sample would be expected to have approximately seven variant copies.…”
We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.
“…The shearing protocol described here is compatible with fast and cheap absorbance measurement [26,27]. If purity ratios indicate that no significant RNA or other contaminant is present, that method can be used.…”
Pipetting and concentration measurement of viscous ultra-high-molecular-weight (UHMW) DNA samples is challenging and often highly imprecise. Effective guidelines for handling UHMW samples are missing in the field. Herein, a simple and low-cost workflow is presented that enables accurate pipetting and reliable concentration measurement. Central to the workflow is the shearing of representative small aliquots of UHMW DNA samples to a fragment size <150 kb by vortexing them for 1 min with a glass bead in a round-bottomed 2-ml tube. Additionally, a solution is provided for accurate quantitation of high-molecular-weight DNA with fluorometric (Qubit [Thermo Fisher Scientific, MA, USA]) methods by using an appropriate genomic DNA standard, resulting in values that match spectrophotometric (Nanodrop [Thermo Fisher Scientific]) optical density readings.
“…is is especially relevant for evaluating the stability of expression of endogenous control genes (housekeeping) [13]. Finally, He et al [43] pointed out the importance of determining the method with which genomic DNA concentration will be evaluated because the 6…”
This study aimed to validate an analytical method to determine DNA concentration using standard reference material (NIST SRM 2372) and Sprague Dawley rat and human DNA. Microvolumes were used to analyse DNA samples. Linearity showed correlation coefficients higher than R ≥ 0.9950, and the precision value was ≤2% CV. Trueness based on bias and the percentage of recovery showed bias values lower than Z-test with a 95% confidence level and a recovery percentage within the range (% Rec = 100% ± 5%), and the stability of the samples was 60 days (2–4°C).
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