Droplet Digital PCR
Droplet Digital PCR (ddPCR) is used to perform a variety of analyses, including absolute quantification of nucleic acid targets, gene expression or rare event (mutation) detection. The Bio-Rad QX200 system uses fluorescence (probe or EvaGreen) and water-oil emulsion droplets, approximately 1 nanoliter in size, to isolate and analyze the targets in individual droplets. This technology provides very sensitive analysis and is well-suited for detection of low frequency targets.
View the service pricing schedule for more information about ddPCR pricing.
Droplet Digital PCR applications
- Detect copy number variations without the use of a standard curve
- Detect rare mutations or sequences- detect one mutant in a background of up to 2,000 wild-type molecules
- Perform gene expression and microRNA analysis
- Perform single cell analysis
- Detect pathogens and analyze the microbiome
- Quantitate NGS libraries and validate NGS results
Instrumentation
The Bio-Rad QX200 ddPCR system consists of a droplet generator to form approximately 20,000 water-oil emulsion droplets and a droplet reader to count the targets in each droplet.
How the ATGC’s ddPCR service works
- Investigators are responsible for purchasing custom or inventoried (validated) assays and the appropriate master mix directly from Bio-Rad: http://www.bio-rad.com/en-us/product/primepcr-pcr-primers-assays-arrays.
- The investigator schedules an instrument run with the ATGC.
- The investigator sets up the Eva Green or TaqMan-style, probe-based reactions in a 96-well plate (Bio-Rad P/N 12001925 preferred) with a total volume of 22ul. If the reactions use EvaGreen as a fluorophore, the reactions must be prepared in a Bio-Rad 96-well PCR plate (P/N 12001925).
- The investigator delivers the reactions in a sealed 96-well plate to the ATGC’s ddPCR service.
- The ATGC generates the droplets from the investigator's reactions.
- Samples are transferred from the droplet generator to a 96-well Bio-Rad plate, sealed and placed in a thermal cycler for PCR.
- Following PCR amplification the samples are placed on the Droplet Reader where the droplets are examined sequentially, similar to a flow cytometer, providing an independent digital measurement. The droplets are scored as either positive or negative for the marker of interest.
- Data Output-Investigators are provided with an analysis file. Further analysis of data can performed, by the investigator, using Bio-Rad’s free QuantaSoft™ Software which is free to download here: http://www.bio-rad.com/en-us/SearchResults?Text=quantasoft&TabName=SOFTWARETYPE
Droplet Digital PCR submission
- Contact Denaha (D.J.) Doss to schedule your instrument run. Please give at least 24 hour notice.
- Download and complete the Service Request and Plate Layout forms and email them to: ddPCRSubmissions@mdanderson.org. You may also contact the ATGC for the forms.
- Provide the reactions in a 96-well plate (Bio-Rad P/N 12001925 preferred), sealed and centrifuged.
- Results can be expected the same day, provided the plate is submitted before noon.
Bio-Rad support for assay design or data analysis
By telephone: 1-800-4-BIORAD (1-800-424-6723)
By email: support@bio-rad.com