ddPCR and qPCR
Digital Droplet PCR
Digital PCR is the latest generation of PCR technology for absolute DNA/RNA target quantification. With digital PCR the absolute quantity of the detection target is reported as the number of copies present in a sample. This determination of absolute copy number is achieved on the basis of presence or absence (binary, digital) of single molecule amplification in a large number of nano-volume PCR reactions. Its high sensitivity and accuracy makes it widely applicable for the following applications:
- Detection of circulating tumor DNA (ctDNA) in noninvasive liquid biopsy
- Identification of rare DNA mutations associated with cancer and other diseases
- Pathogen id. at levels below the current detection limit
- Determination of copy number variants (CNVs)
- Measurement of gene expression changes, especially at finer levels (e.g., < 2-fold)
- Single-cell analysis for precise detection of DNA/RNA targets at ultra-low levels
- Screening of CRISPR genome editing products
- Characterization and quantification of NGS libraries
- Validation of NGS findings
NUSeq maintains a Bio-Rad QX200 droplet digital PCR (ddPCR) system as the Core’s dPCR platform. This system includes a droplet generator, a C1000 Touch thermal cycler, and a droplet reader. From a 20 ul PCR reaction mix (prepared by user), the droplet generator makes 20,000 droplets. Within the dynamic range of 1-120,000 copies target molecule per 20 mL reaction (for human DNA this is up to 400 ng, assuming 1 copy/haploid genome), not all droplets contain the target molecule and the fraction of droplets containing at least one copy of the target is determined by its copy number in the original sample. After undergoing PCR, all droplets are analyzed for presence or absence of target amplification. From the fraction of positive droplets, absolute quantity of the target in the sample in digital format, i.e., # of molecules per mL, is calculated using Poisson statistics.
The QuantaSoft Analysis Pro software used for data analysis is available at the Core and free to download (click on the Download tab). To report target amplification, EvaGreen, or specific probes labeled with fluorophores FAM and/or HEX (or VIC), can be used. Multiplexing is accomplished with the two-color detection feature of the QX200 ddPCR system. With the use of FAM- and VIC-labeled TaqMan probes, 5-plex can be achieved using strategies such as varying concentrations of different probes. To get started on ddPCR, below are some general web resources:
Real-Time Quantitative PCR
NUSeq provides a QuantStudio 7 Flex Real-Time PCR System from Thermo Fisher to meet NU user demands for real-time PCR capabilities. This system can be used for target mRNA or miRNA expression, genotyping (SNP or CNV), etc.
The QuantStudio 7 System is compatible with both TaqMan and SYBR Green chemistries. SNP genotyping and CNV assays are usually based on TaqMan chemistry. For example, in the case of SNP detection, two allele-specific probes carrying different reporter dyes are used to differentiate the alleles via detection of fluorescent signals emitted from the two reporter dyes. For mRNA or miRNA expression profiling, either chemistry may be used.
Three block formats, i.e., 96-well, 384-well plate, and TaqMan array card, are provided to scientists for use on this system. No special tool is required to change the blocks. A quick reference guide for the instrument is available here (Experimental setup starts from p.4). This system can be booked through NUcore via the instructions below.
New users must obtain training on the Bio-Rad QX200 ddPCR or QuantStudio 7 qPCR system prior to first use. Please download a copy of the respective user guide (ddPCR Droplet Generator & Droplet Read and QuantaSoft Software Guide; qPCR Quick Reference Guide) to get familiar with the system. After the training, users prepare reactions and run them independently on the instruments (both are equipment-only services), after reserving the instrument using steps below.
To use the QX200 ddPCR or QuantStudio 7 system, users must sign up for time slots through the NUcore ordering system. For new customers who need to set up an NUcore account to make reservations, please refer to the Account Setup page.
- Open a browser and go to https://nucore.northwestern.edu/facilities
- Click on “Login” at the top right-hand side of the page
- Enter your NetID and password
- Click on “NUSeq”
- Click on the link for the required instrument under “Instruments”
- Select the payment source from the drop-down menu
- Enter the date and start time of your reservation.
- Click “Create”
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