About these references:
This page contains key scientific publications relevant to Qubit Fluorometry.
Each reference includes clickable links to the original publication (via DOI) and
PubMed entries where available. These papers provide the theoretical foundation
and practical context for the laboratory techniques covered in this module.
1. DNA Qualification Workflow for Next Generation Sequencing
Simbolo et al. (2013)
Citation:
Simbolo, M., Gottardi, M., Corbo, V., Fassan, M., Mafficini, A., Malpeli, G., Lawlor, R. T., & Scarpa, A. (2013). DNA qualification workflow for next generation sequencing of histopathological samples. PLOS ONE, 8(6), e62692.
Main Findings:
- NanoDrop consistently reported higher DNA concentrations than Qubit, with limited consistency to qPCR for partially degraded DNA
- For high molecular weight DNA from fresh-frozen samples, NanoDrop measurements were consistent with qPCR
- For degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements
- Recommends sequential combination of NanoDrop and Qubit to assess purity and quantity of dsDNA, respectively
- qPCR used as reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification
Relevance to Course: Fundamental comparison demonstrating why we use both instruments - NanoDrop for purity assessment, Qubit for accurate dsDNA quantification.
2. Comprehensive Three-Method Evaluation
Aranda et al. (2024)
Citation:
Aranda, P. S., Versmessen, N., Van Simaey, L., Negash, A. A., Vandekerckhove, M., Hulpiau, P., Vaneechoutte, M., & Cools, P. (2024). Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts. PLOS ONE, 19(6), e0305650.
Main Findings:
- Both NanoDrop and DeNovix (spectrophotometry) reported DNA concentrations 3-4 times higher than Qubit (fluorometry)
- The ratio of spectrophotometry/Qubit increased with higher A260/280 values (indicating impurities)
- For pure DNA (A260/280 1.7-2.0), the ratio was close to 2; for impure DNA, the ratio was higher
- Qubit is more accurate for quantifying pure dsDNA, while spectrophotometry overestimates due to contaminants
- Spectrophotometric methods (DeNovix and NanoDrop) showed highly consistent results with minimal discrepancies
- A single freeze-thaw cycle produced negligible effects on measured concentrations and purity ratios
- DeNovix demonstrated strongest stability (Spearman correlation of 0.999), followed by NanoDrop (0.81) and Qubit (0.77)
Relevance to Course: Explains why students often see different values between NanoDrop and Qubit - spectrophotometry measures all nucleic acids and contaminants, while Qubit measures only dsDNA. Also reassures students that sample storage and handling (freeze-thaw) don't dramatically affect DNA quantification results.
3. gDNA and Fragmented DNA Analysis
Kumar et al. (2023)
Citation:
Kumar, S., et al. (2023). Comparative analysis of DNA quantification methods for gDNA and fragmented DNA. Analytical and Bioanalytical Chemistry.
Main Findings:
- Qubit consistently reported lower DNA concentrations than NanoDrop, especially for fragmented or impure samples
- Qubit is more specific for double-stranded DNA and less affected by contaminants
- NanoDrop is faster and provides purity ratios but is less accurate for low-concentration or degraded DNA
Relevance to Course: Important for understanding why Qubit is preferred for PCR setup - it measures the actual dsDNA template available for amplification.