Quantitative PCR (qPCR) is a highly sensitive, specific and reproducible method to quantify DNA or cDNA present in low amounts in a sample. It has thus become the go-to technique in this area.
It is necessary that its results be always predictably high in accuracy and that it be always easy to run this test, so that it can be used to quantify the expression levels of any set of genes with regard to each other, to detect low levels of genetic material, or to provide absolute measures of NGS libraries.
The Fluent Automation Workstation with its FluentControl™ software are poised to fill this need for a highly replicable and dependable automated system that needs little operator input, for qPCR.
When this is used in tandem with the KAPA SYBR FAST qPCR Kit, the complete process is automated and work proceeds automatically from one process site to another, resulting in high efficiency of amplification for target amplicons at different levels of GC pairing.
The Fluent system has user-friendly features such as the integrated TouchTools™ user interface (UI) and Smart Commands, that make the workflow easy to understand, while still offering an adaptable set up for this use.
This means the user can set specific parameters for each process run, or instead set up basic protocols with simple inputs such as fixed volumes and other variables, which are predefined so as to make it easy and fast to set up the run.
Another advantage of this system is the accurate level of detection of the liquid level. This lets the user refill liquid reagents as and when necessary in response to the prompts at the UI. Since this can be done even during a run, it allows uninterrupted functioning without having to stop because reagent volume is inadequate; it also avoids wastage of liquid volumes and makes sure that reagent use is optimal.
The TouchTools interface helps the user gain an understanding of the process so that it is easy to adapt the functions to individual laboratory requirements. This includes sample number, master mixes, replication of mixes from a menu, or manual input of components (Figure 1).
Other options include the capability to generate a standard curve using the number of dilution points as a variable. The worktable can be set up with ease because of the provision of instructions on-screen, and the availability of a schematic drawing of the whole process throughout the procedure.
Following the loading of all the equipment that is needed on the worktable the operator approves the set up and the run is automatically begun.
Materials and methods
The protocol for the qPCR procedure in this experiment was set up on a Fluent Automation Workstation with an Air FCA™ having eight channels, which uses single-use filtered 50 µl tips from Tecan, and which runs on FluentControl software (version 1.6).
The KAPA Kit was used for the qPCR in compliance with manufacturing instructions and guidelines with regard to using this kit to explore gene expression, as shown in Tables 1 and 2. The first step used genomic DNA (K562) whose initial concentration was known to be about 100 ng/20 µl reaction, and which was then diluted on the Fluent system to 1:5 in a mixture of 10 M Tris-HCl and 0.1% Tween® 20 buffer.
The process was performed in triplicate on a CFX96 Touch™ Real-Time PCR Detection System, while CFX Manager™ Software, version 3.1(both from Bio-Rad) was used to analyze the obtained data. Two housekeeping genes (RPL13a and ActB) that are frequently used in such studies were subjected to this reaction, and the results were converted to a standard curve from about 100 ng going down to 32 ng per 20 µl reaction.
These genes are amplicons of varying GC content of 52.8 and 60.1%, respectively and differing lengths of 231 and 153 base pairs respectively. Both manual and Fluent-based preparations were made to allow for relative performance to be directly assessed. Manual preparations included pipetting of serial dilutions for data on which the standard curve was based.
The setup of the worktable included the following labware:
- A tube rack which would hold 2 ml microcentrifuge tubes containing 2x KAPA SYBR FAST qPCR Master Mix (which has both primers and undiluted standard DNA)
- A trough of 100 ml capacity containing 10 mM Tris-HCl + 0.1% Tween 20 buffer
- Boxes with 50 µl tips (single-use)
- 3 96 PCR plates for holding the sample, standard and reaction agents
Analysis and results
Two methods were used to analyze the data, a single threshold defined by the user and the baseline subtracted curve fit. Using these, the reaction efficiencies, the R2, the slope of the reaction and the y-intercept were all calculated.
The results of these analyses showed that both manual and automated techniques of qPCR fell within desirable limits, namely, 90-110%, without respect of amplicon GC content and length. Since the pipetting was done with adequate precision so as to give consistent volumes in each of the triplicated procedures, the standard curves were reliable as shown in Figure 2.
This showed that automated qPCR is a method able to withstand minor variations in the setup and yield excellent results within about 25 minutes (for a plate with 96 wells) which is considered to be a short time.
Figure 1. TouchTools interface with on-screen instructions for entering the variables (top) and loading the worktable (middle), plus a schematic overview of the set-up (bottom).
Table 2: Cycling protocol
The experimental yield shows that the Fluent system when used with the KAPA Kit generates a simple automated workflow and process for reliable and reproducible results with qPCR. This was so when the amplicons were different with regard to the GC content, and required little user input.
The advantages include the easy-to-use TouchTools UI which also imparts ease of adaptation, and lets the platform be modified by the user to fit specific situations with respect to different workflows. Thus, operators need not be trained for long to use this platform efficiently. In addition, the MCA arm may be used to pipette through 96 or 384 channels, to further speed up the run if necessary.
Figure 2. Automated vs. manual PCR set-up. Amplification plots for ActB (A) and RPL13a (B) genes obtained after qPCR reaction prepared either on the liquid handling platform (blue) or by manual pipetting (green) show minimal difference. The melting curve profiles (inset in the amplification plots, where y axis: –d(RFU)/dT and x axis: temperature in °C) show a single peak indicating specific amplification of one PCR product. Standard curves generated for ActB (C) and RPL13a (D) in the automated workflow achieved high efficiencies – 95 and 107%, respectively – compared to 97 and 95% using the manual workflow (not shown).
Implementation of the KAPA SYBR FAST qPCR Kit was achieved through close collaboration with Marsha McMakin, Sr. NGS Automation Engineer at Roche.
- Pfaffl, M.W., Horgan, G.W. and Dempfle, L. (2002). Nucl Acids Res 30(9): e36.
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