Digital Droplet PCR

The QX200 Digital Droplet PCR System

In order to use the QX200 system you must first be trained!  You can request training by emailing John Anderson or Carol Wilusz or through iLabs.  Free samples of EvaGreen and Probe Reagents plus cartridges are available until stocks run out.

Current fees are $1.97 per sample plus $2.35 per run effective March 1, 2016.  Your account number and # of samples will be requested when you open the Quantasoft Program to initiate droplet reading.  Droplet generation and PCR are free.

In addition to the full QX200 system (droplet generator, plate sealer, C1000 PCR machine plus droplet reader) located in Biology 462 (Sloan Lab), there are also satellite droplet generator/plate sealer/PCR stations at the locations listed below.  You can set up and run your PCR reactions at these locations and then bring your samples to Microbiology B406 for reading immediately or store for reading at a later time.

Satellite Droplet Generation/PCR stations

MRB Room 479 (Argueso Lab)
Pathology Room 304 (Akkina Lab)
RIC/Foothills D140 (Stenglein Lab)
Plant Sciences E111  (Gaines Lab)
VTH – Diagnostic Medicine Center (MacNeill Lab)

Frequently Asked Questions

Learn More About Digital Droplet PCR

Digital Droplet PCR uses microfluidics to separate a standard PCR reaction into a water/oil emulsion of thousands of nanodroplets, essentially allowing one to perform thousands of independent PCR reactions in a single tube.  Following PCR in a standard thermocycler, the number of positive droplets is determined using  flow cytometry and the number of copies of DNA template in the initial reaction can be inferred using Poisson statistics.  The BioRad QX200 can read droplets in two different fluorescence channels (FAM and HEX) allowing for multiplexing of TaqMan® probes.  The technology is also compatible with EvaGreen dye-based PCR.

Digital droplet PCR has advantages over real-time quantitative PCR in that it allows for easy, sensitive and accurate quantification of template copy number without the need for standard curves.  In addition, due to the large number of droplets in each tube, each of which is an independent assay, there is no need for technical replicates in ddPCR analysis.

Yes!

Briefly, the fluorescence intensity for each product can be varied by:
(i) selecting primers that generate products of different lengths – a 60bp PCR product  and a 300bp product will bind very different amounts of Evagreen giving easily distinguishable products
or (ii) by varying the PCR reaction conditions to reduce the efficiency of one reaction – adding less of one primer set, or designing a primer set that works better at a lower Tm can be very effective.

Here are some references on the topic:

High Sensitivity Detection and Quantitation of DNA Copy Number and Single Nucleotide Variants with Single Color Droplet Digital PCR Miotke et al Anal Chem. 2014; 86(5): 2618–2624.

Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.  McDermott et al  Anal Chem. 2013;85(23):11619-27

The short answer is yes!  Any primer set that works with SYBR-Green should be easily adapted for EvaGreen ddPCR.  In addition, TaqMan® probes that can be detected in the FAM or VIC/HEX channels can also be used with the QX200.

Two things to note are:

(i) The linear range of ddPCR (10-100,000 copies) is lower than for qPCR (100-10^9 copies) and it can therefore be easily overloaded.  It is recommended that you use 10-50x less cDNA template for gene expression assays than you would use in qPCR assays, especially if your template is abundant.
(ii) Genomic DNA may need to be pre-digested.  Often this can be performed in the PCR reaction buffer prior to droplet generation.

HOWEVER if you are designing a new set of primers specifically for ddPCR there are some differences between primer design for ddPCR and for qPCR.  BioRad provides some tips here.

QuantaSoft software is compatible with PCs and is available free of charge.  Request a copy by emailing John Anderson (janderso@colostate.edu) or Carol Wilusz (cwilusz@colostate.edu)

Reservations for the Droplet Generators/PCR and for the Reader, and for training are now made through iLabs.

Contact Carol Wilusz cwilusz@colostate.edu if you have problems accessing iLabs.  You will need a CSU account number and PI assigned to your name.

Guidelines

Please consider the following guidelines when making a reservation:

  • Allow ~ 2hr for Droplet Generation and PCR.
  • Allow 10 min for set up plus 2.5 min per sample for Droplet Reading.
  • You may use the droplet reader computer for analysis only if others are not waiting to use the instrument.
  • You may request a free copy of the QuantaSoft software from Carol Wilusz or John Anderson so you can complete the analysis on your own computer.

The Droplet Reader and a Droplet Generator/PCR machine and plate sealer are located in B406 Microbiology.

DdPCR has the edge over qPCR in several key applications:

  • Copy Number Variation
    DdPCR is much more sensitive for these types of study because it can accurately distinguish 1.2 fold differences in copy number.
  • Quantification of Viral or Bacterial Load
    DdPCR results are given in the form of  absolute copy number of DNA template in the reaction.  Therefore no standard curves are needed which saves money and time.  DdPCR is also optimal for detecting rare events such as very low level infection.
  • Rare Mutation Detection DdPCR is ideal for detecting rare events such as point mutations in cancer and CRISPR/Cas generated mutations in cultured cells.
  • Quantifying small changes in gene expression
    qPCR struggles to accurately quantify small changes (<2-fold) in mRNA abundance.  DdPCR is superior in this case as it can accurately distinguish 1.2 fold differences.
  • Quantifying libraries for Next Generation Sequencing Sequencing libraries must be accurately quantified to ensure optimal cluster density on the flow cell. DdPCR is superior at quantifying the number of functional fragments present in your library.

Check out BioRad’s Application Guide

Currently the MQ Core has free samples and will provide everything needed to perform 8 reactions using either probes or EvaGreen along with training on how to use the droplet generators and droplet reader.

Only reagents and plasticware from BioRad are authorized for use in the QX200 system.  Other reagents, including standard qPCR reagents will damage the instruments.

For EvaGreen PCR

  • EvaGreen ddPCR supermix               – 200rxns BioRad 1864033
  • Cartridges for Droplet Generation     – 24pk BioRad 1864008
  • Gaskets for Droplet Generation          – 24 pk BioRad 1863009
  • Droplet Generator Oil for EvaGreen  – 2 x 7ml BioRad 1864005
  • Twin Tec semi-skirted 96 well plate   – 25 pk Fisher or VWR 951020362
  • Foil plate seals                                         – 100 pk BioRad 1814040

For TaqMan®/Probe-based PCR

  • Probe ddPCR supermix                     – 200 rxns BioRad 1863023
  • Cartridges for Droplet Generation     – 24pk BioRad 1864008
  • Gaskets for Droplet Generation          – 24 pk BioRad 1863009
  • Droplet Generator Oil for Probes        – 2 x 7ml BioRad 1863005
  • Twin Tec semi-skirted 96 well plate   – 25 pk Fisher or VWR 951020362
  • Foil plate seals                                         – 100 pk BioRad 1814040

In addition to the standard reagents shown above, BioRad also offers One Step ddPCR SuperMix for Probes, and ddPCR SuperMix for Probes with dUTP.  Larger packages are also available.

Please note that BioRad requires you to purchase their Probe ddPCR supermix if you purchase their pre-designed probes and primers.

Through December 2015, BioRad is offering 20% discount to CSU customers on ddPCR reagents and plastics.  Contact David Miller (David_Miller@bio-rad.com) or Cammie Austin-Bilo (Cammie_Bilo@bio-rad.com) from BioRad for a quote.

In some ways ddPCR is cheaper than qPCR:

  • There is no need for a standard curve – as the reaction is assessed at the endpoint it does not matter too much whether it is 100% efficient.  This saves you time and money in optimization.
  • There is no need for technical replicates – this saves you time in setting up reactions and reduces the cost of reagents – depending on the size of your reactions this can save $0.80-$1.60 per sample
  • You can multiplex to assay your reference gene and gene of interest in the same well which will halve your expense and pipetting!

However there are additional costs associated with ddPCR:

  • Cost of droplet generation cartridges, gaskets and oil – ~$1.60 per sample
  • Effective March 1, 2016 fees are $1.97 per sample plus $2.35 per run for droplet reading to cover the cost of the service contract and oil, plus a small fee for technical support.  This will come to $18.11 for 8 samples or $191.47 for 96 samples.

Overall:

  • You can expect ddPCR to be slightly more expensive than qPCR for some applications, and cheaper for others (generally those where you always perform a standard curve)
  • However you should take into consideration that ddPCR is more sensitive and will give more reliable results in many applications.

We have not tried this.  You need 10,000 droplets to be able to determine copy number accurately using Poisson statistics.  20μl is routinely giving us 14-19,000 droplets.  So it is possible you can get away with 15μl reactions once you know what you are doing.

Let us know if you try it and it works!

Yes!  If you do not use all the wells at one time, you can go back and reuse empty wells for a second experiment. We have tried this and the seal was still effective second time around.  Be careful to use the recommended plates and seals though.  

If you are going to reuse a 96-well plate then when removing the first seal, be careful not to contaminate the empty wells that you want to use for the next experiment with DNA from the previous experiment!

Unfortunately the microfluidic cartridges are not reuseable primarily because of the potential for contamination and the difficulty in cleaning out the tiny channels.

You probably can reuse the gaskets (although BioRad does not recommend it).  At a minimum, as they are reversible you can flip and use the other side.  We believe you should be able to rinse them in bleach and then water and dry for reuse.   However, the potential for contamination of your samples with DNA from previous experiments is still there.