A: The minimum microdroplet volume of samples is related to your experiment's mutation A: According to the Poisson distribution principle, nucleic acid concentration less than or equal to 5 copies/droplet (100000copies/20ul) can ensure that there are spare microdroplets in the whole body, and the results can be guaranteed to be stable by statistical conversion, it is not necessary to require a single copy per microdroplet. It is not necessary to require a single copy of each microdroplet. 20,000 microdroplets correspond to 100,000 copies of nucleic acid. Even if there is a loss in the total number of droplets, a typical detection limit of 15,000 droplets should be 75,000 copies.
Theoretically, as long as there is 1 negative microdroplet left, ddPCR will be able to calculate the sample content based on the Possion distribution, but the CV% will be very large. An acceptable dynamic range is a CV%<5% sample content range. For any dPCR platform, the optimal sample concentration is the one corresponding to λ=0.16, when the CV% is minimized (~1.5%), which corresponds to the optimal amount of sample. However, in the actual test, the sample concentration within the dynamic range required by the platform is OK
For 20,000 droplets, the upper limit of the sample concentration (theoretically λ=5.5) corresponds to 99.6% of positive droplets and 0.4% of negative droplets, which corresponds to the number of negative droplets is 80. That is, it is OK to have more than 80 negative microdroplets. In fact, theoretically it can be less, but considering other uncertainties, this number is closer to the actual measurement results
A: The number of microdroplets is lower than 9000, statistically speaking, the sampling volume is too small to use Poisson distribution correction, especially for high concentration samples, the quantitative error will be larger, and the detection limit will be reduced. If compound wells are present, since all microdroplets between the compound wells are independent reaction systems, the compound well data can be combined and analyzed, treating multiple wells as one.
For the QX200 system, if the reaction volume is 20 microliters, then the number of droplets generated is around 20,000. The actual detection of less than 20,000 often stems from the dead volume of the system, losses during microdroplet transfer, and other uncertainties. In other words, after the microdroplet preparation, the target nucleic acid has been randomly distributed in 20,000 microdroplets, and the percentage of positive microdroplets has been established, so the subsequent analysis of less than 20,000 microdroplets will have no effect on the quantitative results (samples of suitable concentration). In fact, we have also encountered cases where the total number of droplets in different batches of the same sample ranged from 4,000 to 20,000, but there was no difference in the quantitative results. Note that the premise of the above statement is "the right concentration of the sample", if the sample concentration is too high or too low, the total number of microdroplets analyzed is low, which will increase the subsampling error of another link, and the accuracy and precision of quantification will be affected. Therefore, the definition of 8000 microdroplets is based on the quality control point of view on the one hand. If you don't reach so many microdroplets, there must be a problem, and it is recommended to redo it; on the other hand, for the samples with ultra-high or ultra-low concentration, the error brought by 8000 microdroplets is still smaller than that brought by the sampling error of the samples themselves.
A: Protein, organic solvents, surfactants, high-viscosity substances in the sample, the ambient temperature when generating microdroplets, and factors such as PCR will affect the number of microdroplets. The main judgment in the blood sample is the protein residue and the alcohol that is not removed during the extraction process.
A: Primer probes are usually dissolved in water or TE, and account for a very small percentage of the entire 20 microliter reaction system, so the impact on the ionic strength or pH of the final reaction system is extremely low. And ddPCR reaction premix itself is a buffer, can maintain a relatively stable pH. samples are generally TE or pure water elution, if there is no problem with the extraction of the system has basically no effect, the only possible situation is that the extremely high salt ion concentration increases the osmotic pressure of the micro-droplet affecting the internal and external phase equilibrium, but the general isotonic solution for the general human blood, there will not be such a high concentration of salt, unless there is a problem with the extraction kit. The high ion concentration in the sample cannot be washed out when concentrating the sample through the column. The effect of ions on microdroplets is minimal compared to the proteins, organic solvents, surfactants, and high viscosity substances in the sample.
A: A droplet is considered stable when the final number of active droplets is greater than 10,000 and there is no step in droplet height in the 1D plot.
A: The validity of ddPCR experiments needs to be differentiated in several ways:
Theoretical sensitivity is 1 copy of nucleic acid. The actual sensitivity needs to be analyzed for your different assays and different extraction methods, and the reproducibility is also different in different concentration ranges of different assays. You will need to refer to the literature, your own experiments, or validation data from FDA medical device registrations.
For a PCR instrument is generally rarely used to analyze the sensitivity of the statement, because each different sequence can be judged as a different analyte, the same method for different analytes LLD, BLD, AS, FS are different, need to be accurate to the direction of the application of different applications and application of reagents.
A: Bio-Rad's different ddPCR supermixes produce different microdroplet volumes, which is why it is important to correctly select the corresponding supermix type in the software during setup experiments, and the software will automatically call up the microdroplet volumes of the corresponding reagents for calculation. The droplet volumes of different reagents have been tested and applied in the calculation of quantasoft software
A: 1. insufficient number of droplets in a single well, 2. insufficient sample volume in a single well, 3. need to calculate the mean and confidence interval between technical replicates. If there are perform technical replicates in the experiment, it is usually recommended to merge the microtitle of each technical replicate to analyze, which can improve the accuracy of the data
A: In double ddPCR experiments, if there is interference or competition between the two replicates (which is usually manifested by the fact that the four clusters of droplets can't be in the rectangle of the line), then the use of noose to divide the microtitle will be more accurate. (Examples can be discussed on site if available)
These two quantitative results are not really contradictory. However, for mutation detection assays developed by ddPCR, whether to use copy number or mutation rate as LOD is still open to discussion. For Liquid biopsy, it may be more useful to analyze both assays at the same time. Personally, I prefer to use copy number concentration, and wild-type results are essential for IPC. the determination of mutation percentage may be affected by sample preparation.
Corrections are required to compensate if new fluorescent markers are added or if the background fluorescence signal of a negative microdroplet is negative.