The MCIC acquired the Bio-Rad QX200 Droplet Digital PCR system.
Digital PCR (dPCR) is quickly becoming the go-to complementary technology to the traditional real-time quantitative PCR (qPCR). Both, qPCR and dPCR provide sensitive and robust detection and quantitation of nucleic acids using fluorescence, and each one of these methods has distinct advantage. ......
Quantitative PCR measures overall sample fluorescence at each amplification cycle, which is then used to calculate the relative nucleic acid amounts in the initial sample. Digital PCR gives an absolute nucleic acid quantification. Using microfluidics or oil droplets, the PCR reaction is partitioned into thousands of nanoliter-size aliquots, where separate PCR reactions occur. Fluorescence of aliquots with a positive PCR reactions is measured and the average number of molecules per aliquot is estimates using Poisson distribution and then converted into molar concentration by dividing by the aliquots volume (https://www.nature.com/articles/s41598-017-09183-4).
Traditional qPCR provides wider dynamic range, high throughput and is economical for screening very large numbers of samples as reactions can be performed in 384 well or larger microtiter plate formats. On the other hand, dPCR offers higher sensitivity and can detect changes as small as 10% compared to a twofold change of the qPCR, it quantifies rare targets in complex mixtures, has high tolerance to PCR inhibitors often present in samples such as plants and soil, and it provides rapid and absolute quantification that does not rely on a standard curve. Currently, reactions can be run up to a 96-well plate format.
The higher sensitivity and absolute quantifications make dPCR more suitable than qPCR for the detection of mutations such as SNPs and small indels, for copy number variation, standard validation and haplotyping and for validation of gene expression data from RNAseq experiments.
More information regarding the Bio-Rad QX200 ddPCR site, and detailed information regarding how to set up experiments can be found in the Bio-Rad ddPCR application guide.
The list of all the molecular biology equipment available at the MCIC is found at https://mcic.osu.edu/genomics/equipment-available-to-users.
If you would like to use our droplet dPCR, please contact the MCIC staff.