Automated 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) automated digital Polymerase Chain Reaction (dPCR) with all the necessary proponents and software 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 digital PCR system facilitates the high-throughput precision detection and quantification of specific nucleic acid sequences within a sample. A dPCR process is based on combining DNA samples with primers, probe dyes and PCR reagents. Nucleic acid samples are separated into thousands fixed volume partitions, each of which serves as an independent PCR reactions. Target sequences undergo amplification following a standard PCR principles and are identified via fluorescence detection of non-specific flourophores or sequences specific probes.  A dPCR system is a workhorse equipment in many molecular biology labs that provides absolute quantification of of target DNA sequences within a sample 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.  We request an automated digital PRC system to 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).  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 digital PCR system (Qty 1):  An automated generator partions nucleic acid liquid samples in thousands of nanolitre-sized partitions accurately and quickly, and removes the need for human pipetting. Full automation simplifies the dPCR workflow, minimizes hands-on input, and eliminates user-to-user variability during partitioning. 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 dPCR system detects discrete, volumetrically defined DNA-contaning partitions and quantifies the nucleic acid sequences based on positive/negative fluorescence detection and fluorescence intensity within each individual partition. The the number of partitions exhibiting positive flourescence 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.  Furthermore, dPCR systems may also determine the flourescence intensity within each partition to inform the researcher of sequence specificity ensuring greater accuracy when quantifying the target sequence.  

An automated dPCR system enhances the dPCR workflow by reducing human error during pipetting and partitioning, which is the main cause for variation between samples in a dPCR workflow. Our facility consistently reports high usage of a primary dPCR setup and organizes equipment specific dPCR 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, meidicine and indus

Mandatory Requirements:

1. Digital PCR system
1.1 The PCR system must be able to convert a sample into a minimum of 20,000 partitions.
1.2 The PCR system must be able to process 96 samples in a single run.
1.3 The system must be compatible with a standard 96-well plates for PCR amplification.
1.4 The PCR system must allow for the excitation and detection of a minimum of six fluorophores (six detection channels) and allow multiplexing up to 12 targets per sample.
1.5 The PCR system must have a linear dynamic range of at least five orders of magnitude for the detection of input samples
1.6 The PCR system must be able to process 96 samples, partitioned at a minimum of 20,000 partitions per sample, as specified in p. 1.1 and 1.2 above, in 4 hours or less.  
1.7 The PCR system must be able to encapsulate whole cells to allow precise and accurate cell counts of an edited or modified cell population.
1.8 The PCR system must be able to partition the samples and analyze the partitions following PCR amplification.
1.9 The PCR system must conduct an absolute quantification of the target molecules without the need for a standard curve.
1.10 The PCR system must include a partitions generator, a partition reader and the appropriate software.
1.11 The PCR system must detect the fluorescence of each partition individually, eliminating the need for image optimization.
1.12 As the PCR system will be housed in a core facility and used by many investigators from various programs, the PCR system must not require the use of specialized liquid-handling equipment or robotic liquid handlers that would typically require specialized training and a dedicated technician to operate.

2.Software
2.1 The PCR system must include software for instrument control and analysis.
2.2 The software must be capable of displaying fluorescence measurements by partition.
2.3 The system software must be capable of calculating and reporting absolute concentration, copy number change and ratio analysis for gene expression analysis.
2.4 The software must be capable of calculating the concentration (copies/μL) for each sample and analyze the copy number variation.
2.5 The software must be free of annual costs (e.g., perpetual license, freeware, or open source) and be available for installation on as many computers as needed. 

3. Other Requirements
3.1 Must include delivery, on-site installation, and training.
3.2 Must be capable of providing up to a total of 5 years of annual preventative maintenance visits.
3.3 Must be capable of providing up to a total of 5 years of full mail-in or onsite warranty on all parts.  Coverage must include unlimited repairs inclusive of shipping/travel, parts, and labour (computer exempt).
3.4 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
3.5 Must include a computer with Wi-Fi and ethernet connectivity, compatible with Windows 10 or 11 operating systems, possessing a USB flash port, 1 TB hard drive, and 32 GB of RAM.
3.6 Assembled equipment must fit on lab bench, without overhang, and with sufficient space remaining between the unit and the backwall to provide adequate ventilation; final assembled dimensions with allowances for ventilation must not exceed 13’ (foot) W x 29” (inch) D x 30” (inch) H.
3.7 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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