Automated Droplet Digital PCR for Absolute Quantification of Gene Expression

The Unversity of Ottawa is pleased to invite qualified proponents to submit a proposal for the provision of one (1) Droplet digital polymerase chain Reaction (ddPCR) system with automated droplet generator, one (1) PCR plate sealer, and one (1) thermal cycler. Together, these components comprise a full droplet digital PCR (ddPCR) system for absolute quantification of gene expression.

The University’s research program will use this equipment for absolute quantification of gene expression across a range of biological samples. An automated droplet digital PCR system facilitates the high-throughput precision detection and quantification of specific nucleic acid sequences within a sample. A ddPCR process is based on combining DNA samples with primers, probe dyes and PCR reagents. A droplet generator partitions liquid nucleic acid samples into thousands of nanoliter-sized droplets, each of which is measured as an independent PCR reaction. Samples are transferred and sealed on 96-well plates, PCR-amplified using a thermal cycler, and processed on the droplet reader to detect and quantify nucleic acid sequences. A ddPCR system is a workhorse equipment in many molecular biology labs that provides an absolute count of the number of target DNA sequences for applications that include mutation detection, gene expression analysis, genome edit detection, copy number variation studies, multiplex mutation screening, viral titer measurement, contamination testing, and research into infectious diseases.  The automated droplet generator and complete droplet digital PRC system will be housed in the Core Molecular Biology and Genomics Laboratory (CMBGL) at the University of Ottawa. This equipment will accommodate the experimental needs of more than 36 research groups across several Faculties at the University (>100 users) and we require a high level of automation, and ease of usage to increase the capacity of the facility for various experiments. Finally, this equipment will be managed by a trained technician that will ensure accessibility to HQP from various labs, Departments and Faculties across uOttawa, as well as users external to the University.

1. General Description:

An automated droplet generator for a droplet digital PCR system (Qty 1):  An automated droplet generator partions nucleic acid liquid samples in thousands of nanolitre-sized droplets accurately and quickly, and removes the need for human pipetting. Full automation simplifies the ddPCR workflow, minimizes hands-on input, and eliminates user-to-user variability during droplet generation. This ensures the most consistent results within biological and technical replicates, and reduces user errors that could lead to equipment damages and experimenal flaws. The droplet reader recieves discrete, volumetrically defined DNA-containing droplets in a single file line and quantifies the nucleic acid sequences based on fluorescence. The intensity of the signal is directly proportional to the amount of DNA molecules present in the sample, thus providing an absolute count of the number of target DNA sequence(s) in the sample.

PCR plate sealer (Qty 1):  A 96-well plate sealer is semi-automated heat sealer that provides consistent and uniform sealing across an entire microplate to prevent sample evaporation from the PCR workflow.  This equipment is benefitial for all PCR-based workflows and any experiments where plate sealing is required.

Thermal cycler (Qty 1):  A thermal cycler is required to carry out DNA amplificaiton through PCR. Thermal cyclers offer precision modulation of temperatures as a function of time across sample wells to create a controled environment for melting DNA, annealing primers, and elongation of the coding strand. Controled repeating of this process is required for exponention amplification of sequence specific regions of DNA.\

Together, this equipment will facilitate a complete ddPCR workflow and reduce human error by automating the droplet generating process. Our facility consistently reports high usage of a primary ddPCR setup and organizes ddPCR information seminars with high attendance rates, emphasizing the demand for this equipment.  As a workhorse equipement in the CMBGL, we expect a long life span as well as reliability. The output of using the infrastructue is high-quality scientific results that are to be published in peer-reviewed journals and as conference abstracts. The equipement will be used to provide multiple HQP (>100) hands-on training in many areas of molecular biology important in academia, medicine and industry.

Mandatory Requirements:

1. Droplet digital PCR system with and automated droplet generator:
1.1 The system must include a droplet generator, a droplet reader and appropriate software
1.2 The system must be able to convert a 20 μl sample into a minimum of 20,000 nanolitre-sized partitioned droplets
1.3 The system must be compatible with standard 96-well plates for PCR amplification
1.4 The system must allow for the excitation and detection of a minimum of six fluorophores (six detection channels) per droplet (FAM, HEX (or Vic), Cy5, Cy5.5, ROX, ATTO 590 Dyes)
1.5 The droplet reader must have the ability to automatically detect fluorescence from all channels during the reading process
1.6 The system must have a linear dynamic range of at least five orders of magnitude for the detection of input samples
1.7 The system must be able to achieve precision of at least ±10%
1.8 The system must conduct an absolute quantification of the target molecules without the need for a standard curve
1.9 The system must detect the fluorescence of each partition individually, which eliminates the necessity of image optimization
1.10 The system must be compatible with the thermal cycle temperature gradient features for optimization of the thermal cycling protocol
1.11 The system must be able to detect droplets without the need for additional passive dyes
1.12 The system must include software for instrument control and analysis that can be installed on a minimum of three computers
1.13 The software must be free of annual costs, e.g., perpetual license, freeware, or open source
1.14 The software must display fluorescence measurements by partition
1.15 The software must be capable of calculating and reporting results such as absolute concentration, copy number change and ratio analysis (for gene expression analysis)
1.16 The software must calculate the concentration (copies/ml) for each sample and analyze the copy number variation
1.17 The software must allow the data to be exported as a Coma Separated Values (CSV) file
1.18 The automated droplet generator must offer a user-definable layout with increments of 8 wells
1.19 The automated droplet generator must be in a HEPA-filter enclosure
1.20 The system must automatically detect signals at interrogation points and measure multiple criteria including droplet dimensions and proximity between droplets to exclude poor quality droplets.
1.21 Must be capable of providing up to 4 years of annual preventative maintenance visits

2. PCR Plate Sealer:
2.1 Must have a heat time < 3 min
2.2 Must have a temperature range of 100°C to 190°C
2.3 Must include sealing frame
2.4 Must include plate support block that accommodate 96-well plates

3. Thermal Cycler:
3.1 Must have adjustable heated lid that is programmable by temperature and sealing duration.
3.2 Must be programable with a thermal gradient 
3.3 Must have a 96-deep well reaction module
3.4 Must accommodate interchangeable modules for dual PCR reactions and RT-PCR reactions

4. Other Requirements:
4.1 Must include delivery, on-site installation, and training
4.2 Must be capable of providing up to 4-years of full mail-in (or onsite) extended coverage plans on all parts.  Coverage must include unlimited repairs inclusive of shipping/travel, parts, and labour (laptop exempt)
4.3 Packaged equipment must slide into, fit in, and slide out of an elevator with the following dimensions, without obstructing door closure:
            door 47” W X 84” H
            length of 96” door to door
4.4 Must include a computer with Wi-Fi and ethernet connectivity, compatible with Windows 10 or newer operating systems, and possessing a minimum of a USB flash port, 1 TB hard drive and 32 GB of RAM
4.5 Assembled equipment must fit on a lab bench, without overhang, and with sufficient space remaining between the unit and the backwall to provide adequate ventilation. Final assembled dimensions must not exceed 13’ (foot) W x 29” (inch) D x 30” (inch) H
4.6 All electrical components must have CSA (Canadian Standards Association) certification, or accepted equivalent as per Electrical Safety Authority Recognized Certification Mark at the following link: https://esasafe.com/electrical-products/recognized-certification-marks/ Otherwise, all electrical components must be able to undergo the applicable electrical inspection, and/or, certification, without voiding the system warranty.”

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