Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data

Abstract

Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.

A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

“…Here, sampling error may play a role since mutated cfDNA molecules might not be present in each replicate . Therefore, samples should always be analyzed in triplicates if low allele frequencies are expected …”

“…120 Therefore, samples should always be analyzed in triplicates if low allele frequencies are expected. [131][132][133] Although dPCR and ddPCR are cheaper and have lower turnaround times than NGS-based approaches when testing for single mutations only, the main disadvantages are that mutations have to be tested sequentially and that detection of previously unknown mutations is impossible. 118 As all dPCR technologies require only low quantities of template molecules, single molecule detection is advantageous for the analysis of limited amount of cfDNA alleles in plasma.…”

Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia

“…Droplet digital polymerase chain reaction (ddPCR) is a relatively new technology used to identify MRD [68]. Like RQ-PCR, ddPCR uses Taq polymerase in DNA amplification techniques, and fluorescent probes are used to target DNA sequences in a sample; however, RQ-PCR provides only relative quantification, while dPCR provides absolute quantification of target DNA samples [69]. In ddPCR, reaction mixes are partitioned into approximately 20,000 droplets into separate reaction chambers and amplified [70].…”

Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression

“…The total number of droplets detected by each dd-PCR reaction system must equal or exceed 12 000, and the final results were expressed as copies/μL. 15,16 When the total number of positive droplets ≥3, dd-PCR results shall be deemed positive. The above experimental procedures were repeated thrice, with a vice pore used each time.…”