dPCR Protocol

Quantitative PCR Protocols

Although quantitative PCR uses the same basic concept as traditional PCR, the reactions differ in that the amplicons are generally smaller and are detected indirectly using an additional dye or labeled probe or primer.

dPCR is a relatively new technology and each platform and application has specific requirements (see Digital PCR for further details). The protocols are specific for each system and are provided, with excellent support, by the instrument manufacturers. Therefore, the information provided below is a starting point from which specific assays can be developed or optimized.

Bio-Rad QX100 System

The Bio-Rad digital droplet system is based on oil emulsification technology and uses a standard 96-well plate format. Each sample is partitioned into 20,000 reactions, yielding a total of 1.9 million reactions per plate. Reactions are set up using Dual-Labeled Probes containing a 5’ fluorescent label and a 3’ quencher, standard to qPCR reactions. The droplet reader is capable of reading end-point fluorescent signals in the FAM and HEX channels allowing reactions to be multiplexed with 2 targets per sample.

Requirements

  • QX100 Droplet Generator (186-3002; Bio-Rad)
  • QX100 Droplet Reader (186-3003; Bio-Rad)
  • C100 Touch Thermal Cycler with 96-well Fast Reaction Module (185-1196; Bio-Rad)
  • Semi skirted 96-well PCR plate (Eppendorf Cat# 951020362)
  • Bio-Rad 2× ddPCR supermix (186-3010; Bio-Rad)
  • Droplet generation oil (186-3005; Bio-Rad)
  • Cartridge holder (186-3051; Bio-Rad)
  • 8-chamber cartridges (186-3008; Bio-Rad)
  • Rubber gaskets (placed over cartridge to create vacuum seal; 186-3009; Bio-Rad)
  • Pierceable foil heat seal (181-4040; Bio-Rad)
  • Primers and probes(100 μM stocks): Available from sigma.com/oligos

Sample Requirements

  • Template Quality: Template should be high quality purified gDNA free from inhibitors. It is recommended to digest template gDNA with appropriate restriction endonucleases (1 μg DNA/40 μL reactions) and heat inactivate enzymes after digestion at 65 °C for 20 minutes. Following heat inactivation, the template DNA should be diluted at least 7.5× to ensure proper template partitioning.
  • Template Quantity: The amount of template to be included in each experiment depends on the relative abundance of the target gene and the amount of droplets partitioned. The Bio-Rad ddPCR system partitions reactions into 20,000 droplets and it is therefore recommended to use 100 ng gDNA in a 20 μL reaction when the target gene is represented by a single copy. It may be necessary to reduce input DNA in cases where the target gene exceeds 8 copies per diploid sample.
    http://www.bio-rad.com/webroot/web/pdf/lsr/literature/bulletin_6277.pdf

Standard Protocol

A 20× primer/probe mix is prepared as described below. The standard ddPCR master mix is a 25 μL mix that includes the aforementioned primer/probe mix, template DNA and 2× ddPCR super mix.

Table P7-17. dPCR Primer and Probe Mix.

20× Primer/Probe Mix Volume (μL) per 100 μL
100 μM F1 primer 10
100 μM R1 primer 10
100 μM labeled probe 5
PCR grade water 75

 

Table P7-18. dPCR Reaction Master Mix.

Reagent Volume (μL) per Single
25 μL Reaction
2× ddPCR super mix 12.5
20× Primer/Probe Mix 1.25
Template (100 ng/μL) 1
PCR grade water 10.25

 

Samples are loaded into an 8 chamber cartridge using 20 μL of the prepared qPCR sample followed by 70 μL of droplet generation oil in the adjacent wells. A rubber gasket is stretched across the top of the chambers to ensure a vacuum seal. Each 8 chamber cartridge is loaded onto the QX100 droplet generator producing 20,000 droplets per sample. Using a 50 μL multichannel pipette, 40 μL of the generated droplets are transferred to a 96-well plate and heat sealed with pierceable foil. The plate is placed in a thermal cycler using standard 2-step qPCR thermal cycling conditions with a 50% (3 °C/sec) ramp rate. Prior to running thermal cycling conditions, it is recommended to test new primer/probe sets using a temperature gradient to optimize the anneal/extend temperature.

Table P7-19. dPCR Cycling Conditions.

dPCR Cycling Conditions Temp (°C) Time (sec)
Initial Hot Start/denaturation 95 600
Steps 1–2 are repeated through 40 cycles    
Step 1 94 30
Step 2
60 60
Step 3
98 600
Step 4
12 infinity

 

Following thermal cycling, the plate is loaded onto the QX100 droplet reader and end-point reactions are analyzed using Quantasoft™ software.

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/bulletin_6277.pdf

Materials