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Digital PCR

Digital PCRIn collaboration with ARUP Laboratories, the Molecular Diagnostics Section of the BMP Core is now offering services on the RainDance Technologies, RainDrop® Digital PCR Platform

About RainDrop Digital PCR

The RainDance Technologies RainDrop platform is an integrated digital PCR and NGS enrichment system that can be used for a variety of applications, which require increased sensitivity and precision compared to standard real-time PCR.  The RainDrop system generates up to 10 million picoliter-sized droplets per well. Since each droplet encapsulates a single molecule, the absolute number of droplets containing the specific target of interest can be quickly determined. Each droplet contains PCR master mix, primers/probes, and either 0 or 1 copy of template. After endpoint PCR amplification, the droplets are analyzed using a two color microfluidic flow cytometer.

Applications

  • Low Frequency Tumor allele detection (<0.1% frequency)
  • Liquid Biopsy
  • Gene expression
  • Copy Number Variation Analysis
  • Targeted Amplicon Illumina Library Preparation
  • Pathogen detection
  • Target enrichment of DNA sample

Sample Preparation

DNA:

  • Use a commercial spin column based kit for extraction since minor contaminants can inhibit the PCR
  • 10 ng up to 1 µg of DNA (Measured by Qubit)
  • Increasing input DNA increases assay sensitivity, but input may need to be optimized to obtain <10% droplet occupancy.
  • High quality intact DNA from cell lines, blood, fresh/frozen tissue etc, will need to be fragmented on the Covaris and concentrated prior to analysis. The Core can fragment your sample for an additional fee.
  • Fragment DNA samples from FFPE or circulating free DNA do not need to be sheared

RNA:

  • Use a commercial spin column based kit for extraction since minor contaminants can inhibit the reverse transcription step
  • Use a commercial kit for cDNA synthesis, such as the Invitrogen Super Script II system
  • The amount of cDNA input will vary based upon the abundance of the transcript and may need to be optimized to obtain <10% droplet occupancy.

Assay Design

The system works with most two-color (VIC and FAM) TaqMan real-time PCR assays.  The RainDrop system is not compatible with SYBR Green. EvaGreen can be used as an alternative for dye-based dPCR detection, gene expression analysis, or genotyping studies. Prior to running the RainDrop, we recommend optimizing your PCR primers and probe concentrations using a standard real-timePCR instrument.  Alternatively, please contact us if you would like a quote for the Core to design an assay.

Data Analysis

Data is analyzed using the RainDrop Analyst II software. The software reads the positive and negative droplets in each sample and plots the fluorescence droplet by droplet. The fraction of positive droplets in a sample determines the concentration of target molecules. The Core staff will perform an initial analysis of the data and upload it to GNomEx.  For advanced analysis the end user can download RainDrop Analyst II software from RainDance Technologies. 

Streamline Workflow

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