Application of Droplet Digital PCR Technology in Muscular Dystrophies Research
Abstract
:1. Introduction
2. ddPCR Technology
3. Advantages and Disadvantages of ddPCR
4. Hallmarks of a Well-Designed ddPCR Assay
- (1)
- Nucleic acid samples concentration. As well as other molecular biology techniques, the quality, and the quantity of nucleic acid samples, may affect the result and are essential for the accuracy of the assay. No special requirements are necessary regarding the sample preparation. However, it should be noted that some methods of nucleic acid isolation may interfere with the generation of droplets [38] and, therefore, the method must be chosen which offers a good separation of positive and negative droplets as well as the best signal intensity. Qubit (Invitrogen, Waltham, USA) and Nanodrop (Thermo Scientific, Waltham, USA) measurement of nucleic acid samples concentration and purity is essential to achieve reliable results [39].
- (2)
- The design of primers and probes are among the most critical factors for the success of the experiment and should be carefully done to avoid self-annealing or cross-reactivity. Whether or not a design program is used, for primers and probes, the same rules as for qPCR analysis must be apply.
- (3)
- Assay optimization. Achieving accurate interpretable results requires a number of important factors to be considered when optimizing a ddPCR assay. The annealing temperature must be optimized for each target using a gradient PCR range between 55 and 65 °C, an interval in which most targets have an optimal temperature. A temperature is optimized when the largest separation between positive and negative droplets is achieved [37,41].
- (4)
- Controls. In ddPCR technology, the use of a reference gene is not mandatory because of the absolute quantification of the number of targets from a sample. Furthermore, the assays can be affected by technical problems associated with the reverse transcription step. Primer dimers and secondary structures are avoided, and the annealing temperature can be used for reaction optimization. An important aspect of the ddPCR assay is represented by the appropriate use of a specific set of controls that are important for method performance [46,47]:
- (i)
- negative controls—for monitoring a false-positive reaction, which may be a marker of contamination or a poor design of primers/probes, and for the determination of limit of detection (LoD);
- (ii)
- positive controls—useful to test for whether the template amplification occurs under the established reaction conditions;
- (iii)
- non-template controls (NTCs)—for control of contamination in all reagents [48]. Poor design optimization can lead to a bad assay performance.
5. Applications of ddPCR in Muscular Dystrophy Research
5.1. Absolute Quantification
5.2. Copy Number Variation
5.3. Gene Expression and miRNA Quantification
5.4. Non-Invasive Prenatal Diagnosis
6. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strengths | Similarities | Differences | ||
---|---|---|---|---|
qPCR | ddPCR | ddPCR/qPCR | qPCR | ddPCR |
Gold standard technique for target DNA quantitation and gene expression analysis | High precision quantification at low input copy number sequence in a complex background | Both methods have multiplex capability | Relative measurement | Absolute measurement |
Economic costs | High sensitivity | Both methods are easy to use. | Standard curves needed | No need for calibration or standard curves |
Rapid test results | Independent analysis and data processing of samples | Quantification of the amount of target in a certain sample | No sample partitioning | The sample is partitioned into a large number of individual reactions |
High tolerance to PCR inhibitor | The same components used in the reaction (PCR Master Mix, primers, fluorescent probes (Taqman probs FAM and HEX/VIC) | Real time PCR data acquisition | End point data collection |
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Lambrescu, I.; Popa, A.; Manole, E.; Ceafalan, L.C.; Gaina, G. Application of Droplet Digital PCR Technology in Muscular Dystrophies Research. Int. J. Mol. Sci. 2022, 23, 4802. https://doi.org/10.3390/ijms23094802
Lambrescu I, Popa A, Manole E, Ceafalan LC, Gaina G. Application of Droplet Digital PCR Technology in Muscular Dystrophies Research. International Journal of Molecular Sciences. 2022; 23(9):4802. https://doi.org/10.3390/ijms23094802
Chicago/Turabian StyleLambrescu, Ioana, Alexandra Popa, Emilia Manole, Laura Cristina Ceafalan, and Gisela Gaina. 2022. "Application of Droplet Digital PCR Technology in Muscular Dystrophies Research" International Journal of Molecular Sciences 23, no. 9: 4802. https://doi.org/10.3390/ijms23094802