Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR protocols for mouse studies

• Published in: Methods (San Diego, Calif.) Vol. 191; no. 4; pp. 95 - 106
• Main Authors: Lindner, Loic, Cayrou, Pauline, Jacquot, Sylvie, Birling, Marie-Christine, Herault, Yann, Pavlovic, Guillaume
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2021; Elsevier
• Subjects: Animal biology; animal models; Animals; Biochemistry, Molecular Biology; CRISPR-Cas systems; data collection; DMIQE; DNA; DNA - genetics; Droplet digital PCR (ddPCR); droplets; emulsions; fluorescence; gene editing; genes; Genetically modified animals; guidelines; humans; Life Sciences; Mice; nucleotide sequences; polymerase chain reaction; Quality control; quantitative analysis; Rats; Real-Time Polymerase Chain Reaction; Research reproducibility; RNA; RNA - genetics; RT-ddPCR; Genetically modified animals; RT; RNA; Research reproducibility; GMO; CNV; ddPCR; dMIQE; RT-ddPCR; MIQE; RQS; DNA; RIN; Quality control; cDNA; Droplet digital PCR (ddPCR); DMIQE; droplet digital PCR (ddPCR)
• ISSN: 1046-2023; 1095-9130; 1095-9130
• DOI: 10.1016/j.ymeth.2020.07.004

•Droplet digital PCR for DNA and mRNA target quantification are described.•Molecular tools for validating genetically altered animal models are presented.•The protocol is validated for mouse studies using a large dataset.•Its technical optimizations and limitations are evaluated.•We also present a new procedure for simultaneous DNA and RNA extraction.•Additional recommendations are given for dMIQE guidelines. Droplet digital PCR (ddPCR) is a recent method developed for the quantification of nucleic acids sequences. It is an evolution of PCR methodology incorporating two principal differences: a PCR reaction is performed in thousands of water-oil emulsion droplets and fluorescence is measured at the end of PCR amplification. It leads to the precise and reproducible quantification of DNA and RNA sequences. Here, we present quantitative methods for DNA and RNA analysis using Bio-Rad QX100 or QX200 systems, respectively. The aim of these methods is to provide useful molecular tools for validating genetically altered animal models such as those subject to CRISPR/Cas9 genome editing, as well for expression or CNV studies. A standard procedure for simultaneous DNA and RNA extraction adapted for mouse organs is also described. These methods were initially designed for mouse studies but also work for samples from other species like rat or human. In our lab, thousands of samples and hundreds of target genes from genetically altered lines were examined using these methods. This large dataset was analyzed to evaluate technical optimizations and limitations. Finally, we propose additional recommendations to be included in dMIQE (Minimum information for publication of quantitative digital PCR experiments) guidelines when using ddPCR instruments.