Results and Discussion
Automated qPCR Setup Accuracy and Precision
The accuracy and precision of the qPCR setup was evalu ated based on
standard curve analysis (Figure 3). Amplifca tion efciency is equivalent
between automated (95%) and manual (94%) setup, and both values are
within the acceptable range of 90 – 110%. The coefcient of variation
(R²) obtained for the two liquid handling methods is similar with a
value of 1.00, meeting the minimum requirement of 0.99. For standards on
the automated standard curve, the standard deviation of Cq values
between triplicate data points was less than 0.1, translating to a
coefcient of varia tion less than 0.4%. This indicates that any
quantifcation data obtained is accurate, assuming accurate and precise
library dilution.
Figure 3: Standard curves of manual (A) and automated (B) qPCR setups, as well as a summary of associated data (C).
Automated Sample Dilution Accuracy and Precision
Standard 0 was diluted to 1:10,000 by two consecutive 1:100 dilutions
either manually or with an automated process. The average pM value of
the automated Standard 0 reactions (216 pM) is similar to the average pM
value of the manual reactions (222 pM) showing equivalency of the
automated sample dilution process on the epMotion (Figure 4).
Additionally, the automated process resulted in an estimated Standard 0
concentration of 216 pM, which deviates from the expected value of 200
pM by less than 10%. The low standard deviation (CV of 1.4%) among the
twelve replicates shows high precision and highlights the reliability of
the automated sample dilution process.
Figure 4: Comparison between manual and automated serial dilutions using Standard 0.
Performance was further evaluated using two Illumina NGS libraries
diluted in series (1:10K, 1:100K, 1:1M) with four replicates per data
point. As expected for a 10-fold dilution series, the Delta Cq values
between the different dilution factors were within the acceptable range
of 3.1 to 3.6. The Cq standard deviation of both NGS libraries does not
exceed 0.2% showing high reproducibility (Figure 5).
Figure 5: Comparison of automated serial dilutions for two NGS libraries.
Conclusions
The automation of the KAPA Library Quantifcation Kit on the Eppendorf
epMotion 5075t offers a solution for high throughput quantifcation,
reducing hands-on time and human error with a total runtime of only 28
minutes for the here presented setup. The Eppendorf epMotion liquid han
dling systems have the advantage of having a user friendly interface,
with flexible options for modifying the number of libraries, the number
of qPCR replicates, and the dilution factor to desired experimental
design with a maximum of 72 reactions per plate. Reliable, optimized and
ready-to-use epMotion methods are available for small epMotion mod els
such as the epMotion 5073 and larger sizes such as the epMotion 5075.
The presence of a thermo-module on the worktable keeps the qPCR plate
refrigerated during process ing, and detection of tip and consumable
positions before run start allows reliable processing without incident.
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