Digital PCR Technical Notes and Application Notes

We have a handful of technical notes and application notes for our Digital PCR platform. Read the abstracts and visit our Download Center to get your copy of these PDF’s.

New! Digital PCR Technical Notes

• Recommendations for Developing a Multiplex Digital PCR Assay

  Digital PCR (dPCR) is one of the most sensitive and precise methodologies for quantification of nucleic acids and is the preferred approach for the detection of low frequency mutations in a high wild-type background. For detecting minor allele frequency (MAF) using standard dPCR, a duplex assay is utilized to differentiate between wild-type and a mutant single nucleotide polymorphism (SNP) enabling absolute quantification. Recently, however, many investigators have pushed beyond the two probe experimental design and have developed multiplex assays to assess multiple mutations within a single sample.1,2,3 Multiplex dPCR for sequences located in genes such as EGFR and KRAS that have multiple cancer-relevant SNPs, are the most requested assay designs as the presence of these mutations determine a clinical course of action. The following technical note describes in detail the recommended workflow for optimizing a multiplex dPCR assay for detecting multiple SNPs from a single sample.

• Qualifying and Optimizing Digital PCR Assays using Real-time Quantitative PCR

  This following suggested protocol is from Brian Parkin, M.D., a physician-scientist and Clinical Lecturer of Internal Medicine in the Division of Hematology/Oncology at the University of Michigan. His research focuses on the characterization and prognostic impact of genomic minimal residual disease (MRD) in acute myelogenous leukemia (AML), as well as determining the mechanisms of AML therapy resistance and relapse before and after allogenic stem cell transplantation. Using the steps in this technical note, his lab has been able to rapidly validate many AML-relevant dPCR assays for personalized MRD testing on the RainDrop® Digital PCR platform using qPCR as a first step optimization method.

• Digital PCR Provides Superior Sensitivity for the Detection of Rare Cancer Variants Compared to Real-time Quantitative PCR

  The ability to sensitively detect rare variants such as cancer mutations is becoming more important with the prospect of “liquid biopsy”, which utilizes circulating cell-free DNA (ccfDNA) as a biomarker for disease. Depending on tumor stage, type, and burden, detection of mutations circulating in the blood or other biofluids can be challenging due to the fact that circulating tumor DNA (ctDNA) is typically present at a low fraction in a vast background of wild-type ccfDNA. Often times, mutation frequency observed from ctDNA is at or below 0.1% thereby requiring extremely sensitive detection capability to assess these rare events. Current gold-standard technologies, including quantitative real-time PCR and next-generation sequencing, are limited in their ability to measure mutation frequencies much below 0.1-1%. The following technical note demonstrates the superior performance of digital PCR (dPCR) compared to quantitative real-time PCR (qPCR), particularly in terms of detection sensitivity using two pre-validated assays.

Digital PCR Application Notes

• Multiplexing Assay for Gene Copy Number

  This application note presents the results of a novel 5-plex droplet-based digital PCR assay to identify gene copy variations and SNPs. Assay performance demonstrates accurate quantification and superior reproducibility across a set of samples and a wide range of copy number levels.

• Digital PCR System Quantifies Methylated DNA Biomarkers

  This application note illustrates the RainDrop® Digital PCR System’s capability to quantify small changes in DNA methylation. Highlighted is analysis using Zymo Research’s OneStep qMethyl™ kit with low amounts of tumor DNA in single- and multiplex assay formats.

• Digital PCR Fluid Biopsy for KRAS Mutations

  This application note describes a novel droplet-based digital PCR method for the non-invasive detection of KRAS in plasma of patients with metastatic colorectal cancer. This highly sensitive and specific method has the potential to be employed in multiple applications in the clinic, including diagnosis, cancer recurrence monitoring, and treatment management.

• Multiplexing with RainDrop® Digital PCR System

  The RainDrop® Digital PCR System provides unique capabilities and benefits for counting applications such as rare mutation detection, quantifying copy number variation, DNA methylation assessment, and as a pre-test QC and validation tool for DNA sequencing. The power of digital PCR is extended even further with the novel multiplexing capability made possible with millions of droplets. This application note summarizes the simple approach used to develop multiplex assays and provides examples of important multiplexed assays in various research applications.

• Quantifying MicroRNA Using the RainDrop® System

  Unlike most other potential biomarkers, microRNA molecules appear to be cell type and disease specific, as well as stable and readily accessible. This application note demonstrates the use of RainDrop®Digital PCR System as an exciting new tool for microRNA research.

• High-Precision Chromosomal Copy Number Measurement using RainDrop® Digital PCR System

  This application note discusses using the RainDrop® Digital PCR System to overcome these measurement barriers and precisely measure chromosomal copy number differences. The most extreme case demonstrated is the discernment of affected and unaffected samples with a 99% confidence interval when the affected fraction of a sample makes up <5% of the 10ng starting material.

• Using the RainDrop® Digital PCR System for One-Step RT-dPCR

  One-step RT-dPCR has many advantages over a two-step reverse transcriptase protocol – namely: 1) simpler workflow; 2) reduced variability due to fewer pipetting and purification steps; 3) reduces 3’ and 5’ biases introduced by random oligomers and oligo-dT primers; 4) single RNA molecules converted to cDNA without competition. The RainDrop® Digital PCR System provides absolute quantification, removing the need for a standard curve, and has a broad dynamic range that allows for quantification of both high expressing and low expressing genes in a single well. In this study, four commercially available RT-PCR kits were tested in the RainDrop Digital PCR System: SuperScript® III One-Step RT-PCR System (Life Technologies); AgPath-ID™ One-Step RT-PCR Kit (Life Technologies); qScriptTM XLT One-Step RT-qPCR Toughmix® (Quanta BioSciences); and QIAGEN® OneStep RT-PCR Kit (Qiagen). While all kits performed well, this application note presents experimental data for the SuperScript® III One-Step RT-PCR Kit using its specified protocol only.

• Detecting Latent and Active HIV Using the RainDrop® Digital PCR System

  In this application note, Human Universal RNA, Xeno RNA and de-identified samples from some of our key customers were evaluated to demonstrate the RainDrop’s utility for sensitive, precise multiplex detection of HIV DNA or RNA.

• Using EvaGreen® Dye on the RainDrop® Digital PCR System to Quantify NGS Libraries

  Next Generation Sequencing (NGS) has rapidly grown to enable a wide range of research and genetic analysis. An important step in the sample preparation process is to confirm that libraries or sample pools are diluted to the optimal concentration for flow cell loading prior to Illumina sequencing. Overestimation of library concentration may result in a lower than desired cluster density. Furthermore, underestimation may result in higher than desired cluster density, which can lead to poor cluster resolution. Both scenarios result in suboptimal utilization of sequencing capacity.