Figure 1: epMotion 5073l worktable for qPCR setup
Results and Discussion
The automation of the
qPCR setup using the 10 μL dispensing tool and the epMotion 5073l was
compared to the manual setup. For a direct comparison, the manual and
automated setups were performed on a same day using the same qPCR plate.
Assay Efciency
To
evaluate the dispensing efciency of the epMotion 5073l liquid handling
system equipped with the 10 μL dispensing tool, a very sensitive qPCR
assay, the Kapa SYBR Fast qPCR Kit, has been selected. Performances were
demonstrated by generating a standard concentration curve of Lambda DNA
either manually or by using the epMotion 5073l with the 10 μL
dispensing tool. The curves shown in fgure 2A cor respond to a series of
10-fold dilutions of a target from 106 copies to 10 copies.
Figure 2: Data obtained with the Kapa SYBR Fast qPCR
Kit (Kapa Biosystems, cat # KK4602). Standard curves generated using
threshold (A) and qPCR efciency parameters evaluated (B).
Parameters
used to determine the assay efciency are the dynamic range, the R2
value and the detection limit (fgure 2B). A slope of -3.43 re?ects an
efciency of 96% while a R2 value above 0.9999 indicates the good curve
linearity and provides confdence in correlating two values. Finally, the
assay sensitivity is ensured as 10 copies of Lambda DNA can be
detected. As demonstrated by all parameters evalu ated, assay efciency
is preserved when the qPCR setup is automated on an epMotion equipped
with a 10 μL dispens ing tool.
A second qPCR assay was used to confrm
those results. The Kapa Library Quantifcation kit provides all reagents
needed for the quantifcation of Illumina libraries by qPCR. The
efciency was tested on a standard curve using the six ready-to-use
standards included in the kit. The standards are supplied in a bu?ered
solution to ensure stability of the diluted DNA and to reduce DNA
adsorption to plastic.
As the physical properties of this bu?ered
solution were di?erent from water, adjusting the dispensing parameters
was a prerequisite for accurate pipetting of 0.2 μL DNA stan dards for
this assay. Optimization could be achieved easily by modifcation of the
liquid type options. Results obtained with this second assay, are shown
in fgures 3A and 3B, con frming that, thanks to the 10 μL dispensing
tool, the qPCR assay efciency is preserved when the automated setup
involves handling of very low volumes (0.2 μL).
Figure 3: Data obtained with the Kapa Library
Quantifcation kit (Kapa Biosystems, cat # 4824). Standard curves were
generated using six DNA standards with 0.2 μL (A) and qPCR efciency
parameters were evaluated (B).
Reproducibility
Reproducibility
is a key component of real-time PCR assay reliability. The assay
reproducibility when an epMotion 5073l is used for qPCR setup was
assessed on qPCR assays, using the Kapa SYBR Fast Kit and the Kapa
Library Quantifcation kit, by comparing results generated from 24
positive samples containing a low and high number of DNA copies per qPCR
reaction. As illustrated on fgure 4, whatever the qPCR as say
evaluated, the mean Ct value is very consistent for a defned amount of
target for the automated setups as well as for the manual setups.
Reproducibility of a qPCR involving volume manipulation as small as 0.2
μL is ensured when qPCR setup is automated on an epMotion equipped with
the 10 μL dispensing tool. The coefcient of variation does not exceed
0.50% and 0.89% for high copies number and 1.17% and 1.69% for low copy
numbers for Kapa SYBR Fast qPCR Kit and Kapa Library Quantifcation kit,
respectively.
Figure 4: Reproducibility of Kapa SYBR Fast qPCR kit
(A) and Kapa Library Quantifcation kit (B). Mean intersample Ct value,
standard deviation and coefcient of variation (CV) were calculated for
each plate containing 24 positive samples.
Conclusion
In
this Application Note, the capability of the epMotion 5073l liquid
handling system to automate a complete qPCR assay setup involving very
low volumes such as 0.2 μL was demon strated. The assay efciency and
reproducibility were successfully assessed on two di?erent qPCR assays.
Results
obtained with the automated qPCR setup were highly accurate and
reproducible yielding data similar to a manual preparation.
The qPCR,
a highly sensitive application, was used in this Application Note to
demonstrate the robustness of the 10 μL epMotion dispensing tool.
Dispensing of sub-microliter volumes can be extended to a large variety
of applications requiring handling of small volumes such as protein
applica tions, compound screening or cell-based assays.
These results
clearly indicate that the epMotion automated liquid handling system
equipped with the 10 μL dispensing tool is an excellent solution for
scientists interested in auto mation of miniaturized assays. epMotion
automated work stations provide a high assay reproducibility ensuring
reliable results by reducing human error and by providing an accurate
pipetting performance.
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