Digital PCR Sample Preparation
Droplet Digital PCR
The IIHG Genomics Division provide digital PCR (dPCR) services using the Bio-Rad QX200™ Droplet Digital™ PCR System. Digital PCR is a technology that provides the ability to perform ultrasensitive and absolute quantification of nucleic acid targets. While Droplet Digital PCR (ddPCR) system can use the same Hydrolysis probe (Taqman)- or EvaGreen (SYBR-like)-based assays, it gives the ability to quantify template molecules that may be undetectable using the traditional real-time PCR techniques. Also unlike qPCR, there is not a need to generate a standard curve since ddPCR simply counts the number of target molecules contained in the sample as the results are expressed as copies per microliter.
As stated above, the ddPCR technique is particularly useful for low abundance targets, targets in complex backgrounds, allelic variants (SNPs) and for monitoring of subtle changes in target levels. Each molecule of DNA/RNA contained sample is partitioned into a large number of nanoliter sized droplets prior to amplification (Fig 1). Each droplet becomes an individual PCR reaction. After end-point amplification, fluorescence is detected in the droplets in which target sequence was amplified. These droplets are scored as positive. Droplets not containing the target sequence show little or no fluorescence and are scored as negative. Using the Poisson distribution law, the fraction of positive droplets is converted to the number of molecules in the starting sample, without the need for standard curves (absolute quantification).
Digital PCR Sample Preparation
First time users should consult with the service personnel to obtain best practices for setting up and running a dPCR assay on the BioRad QX200 system.
- Contact the Genomics Division to make an appointment. You should plan on 4-5 hours of time, depending on the number of samples.
- Users are responsible for purchasing ddPCR supermix/master mix and droplet generator oil. Please note that different ddPCR Supermixes and Droplet Generator Oils are used for probe- and Eva green-based assays. BioRad ddPCR supermixes and droplet generator oil can be purchased at the Biomedical Research Store (2nd floor EMRB).
- Prepare your PCR reactions prior to coming to the core.
- Transfer 20 ul of your PCR reaction into the droplet generator cartridge - it is recommended that you prepare 22 to 25 ul PCR reactions to account for void volume.
- Including a negative control is strongly encouraged as it will help the analysis software discriminate positive from negative reactions.
- Bring your prepared PCR reactions AND with your Droplet Generator Oil to the core at your reserved time.
- We will provide you the remaining items needed to complete your project.
Digital PCR Fees
There are 2 fees that are assessed for each project run on the ddPCR system: 1) sample preparation fee and 2) instrument fee.
- The sample preparation fee is based on running sets of 8 samples (the Droplet Generator Cartridge holds 8 samples) and includes the consumables required to process 8 samples.
ddPCR Sample Fee = $40 (per 8 samples) - The instrument fee covers the cost to have the service contract and perform the necessary maintenance runs required to keep the instrument running optimally.
Instrument Fee = $75 (per QX200 scan)
Digital PCR Resources
ddPCR: What is it and how can I use it to reproducibly improve the quantification of low-levels of RNA and DNA? Click here to learn more
Bio-Rad hosts a number of useful resources about ddPCR applications, technical webinars, and Assay Guide.
Bio-Rad Applications & References
- Liquid Biopsy
- Copy Number Variation (CNV) Analysis
- Rare Sequence Detection
- Gene Expression
- Single-Cell Analysis
- Pathogen Detection and Microbiome Analysis
- Next Generation Sequencing (NGS)
Bio-Rad Webinars (Below is a partial list of the webinar titles)
- Ultra-sensitive quantification of genome editing events using Droplet Digital™ PCR (ddPCR™)
- >Digital PCR Tips and Tricks: Assay Design
- Gene Copy Number Variation Analysis
- Enabling High-Resolution Gene Expression and miRNA Analysis with ddPCR™
