在進(jìn)行微量體系的液體操作時，如何避免人為誤差與日間變化，如何保證結(jié)果的一致性提高實驗效率？如果減少冗長枯燥的重復(fù)操作，減少昂貴試劑的使用，提升操作體驗節(jié)省實驗經(jīng)費(fèi)？
本篇應(yīng)用旨在介紹如何用10 μL分液工具實現(xiàn)全自動的微量體積的qPCR反應(yīng)體系構(gòu)建。在每個PCR反應(yīng)體系中，樣品量為0.2 μl，單個反應(yīng)體系總的體積為5 μL。
對于最終的qPCR實驗結(jié)果的評估，通過兩個靈敏的qPCR測定來完成。結(jié)果顯示借助epMotion 5073l系統(tǒng)可成功地完成微量體積的qPCR體系構(gòu)建，其精度與重復(fù)性都十分出色。

The 10 µL Dispensing Tool Ensures Highly Accurate and Precise Sub-microliter Volume
Pipetting on epMotion® 5073l

Eric Gancarek¹, Dominik Schneider 2, Sandrine Hamels¹
¹ Eppendorf Application Technologies S.A., Namur, Belgium
² Eppendorf AG, Hamburg, Germany

Abstract

This Application Note shows the possibility to automate quantitative Polymerase Chain Reaction (qPCR) setup involving small reaction volumes by using the epMotion® 10 µL dispensing tool. For PCR setup, a sample volume of 0.2 µL was used in a total reaction volume of 5 µL. The qPCR performances were evaluated by two sensitive qPCR assays.
It was demonstrated that a low volume qPCR setup can be successfully automated on the epMotion 5073l. Excel lent accuracy and reproducibility were obtained for both assays.
The use of the epMotion workstation is an excellent solu tion to increase the consistency and efciency of small volume dispensing by removing the human and day-to day variability. In addition, researchers will save time and money by eliminating repetitive work and by reducing the use of costly reagents, respectively.

Introduction

Nowadays, laboratory processes are becoming more and more complex driving the need for assay miniaturization. The tendency towards assay miniaturization is present in a lot of different applications. An example is the screening of compounds to discover new drugs in the pharmaceutical industry [1]. Indeed, the ability to perform primary screen ing assays in high-density micro-well plates at volumes of 1–2 μL will accelerate the early stages of drug discovery and reduce costs. Another feld is in molecular biological research using the quantitative real-time Polymerase Chain Reaction (qPCR) technology. However, qPCR is an expen sive technology. One way to decrease the costs is to reduce reagent volumes.
In addition to reducing reagent costs, miniaturization offers many other advantages. In applications such as forensic analysis, the volume of extracted DNA available for qPCR is often limited [2]. A miniaturized assay allows achieving the qPCR even with a low amount of DNA. Another advan tage is the possibility to run more experiments with the same amount of biological sample leading to better result interpretation. Finally, by decreasing the volume, it is possible to increase the number of reactions performed in parallel and reduce the analysis time. In the past, low throughput qPCR systems were common in analytical labs. Currently, they are more and more replaced by 384-well instruments and even higher throughputs.
Despite the advantages, miniaturization asks for dispensing small liquid volumes accurately and precisely. Delivery of sub-microliter volumes is difcult to achieve and is a major obstacle to the implementation of miniaturized assays. One of the issues that all labs are facing is the human error. The smaller the dispensing volume, the greater the operator expertise should be. Additionally, in a miniaturized assay, it is usual to work with 384-well plates which also increases the risk of human error. Lastly, beside the human factor, environmental conditions such as small variations in labora tory temperature and humidity can have a signifcant effect on the correct handling of small volumes.
With so many variables affecting the dispensing process, choosing the proper solution for small volume handling is of the highest importance. One solution, especially when a large sample number is required to be processed in a short time, is automation. The capability of the epMotion liquid handling workstation to automate a qPCR assay from Master Mix preparation to 96-well PCR plate setup was already demonstrated [3-5]. By reducing human in tervention and thanks to an accurate pipetting system, epMotion automated liquid handling systems provide high assay reproducibility without cross-contamination, ensuring reliable results.
The new 10 µL dispensing tool developed for the epMotion automated liquid handling systems allows the dispensing of volume as low as 200 nanoliter. The complete volume range covered by all epMotion dispensing tools is now extended from 0.2 µL to 1000 µL. The use of this new accessory en sures accurate and precise automated dispensing of sub microliter volumes. The purpose of this Application Note is to demonstrate the efciency of the 10 µL tool in a qPCR setup by using sample volumes as low as 200 nanoliter.

Materials and Methods

qPCR
> Eppendorf twin.tec® real-time PCR plates 96, semi-skirted(Eppendorf, cat # 0030 132.505)
> DNA LoBind Tubes, 1.5 mL(Eppendorf, cat # 0030 108.051)
> HeatSealing PCR Film(Eppendorf, cat # 0030 127.838)
> HeatSealer S200(Eppendorf, cat # 5392 000.005)
> Mastercycler® ep realplex (Eppendorf)
> Water, Sterile, Nuclease-free, Biotechnology Grade(VWR®, cat # E476)
qPCR Assay 1
> Kapa SYBR® Fast qPCR Kit(Kapa Biosystems, cat # KK4602)
> Specifc primers (Eurogentec®)
> Lambda Phage DNA (Sigma-Aldrich®, cat # D3779)
Real-time qPCR assay developed for Lambda phage DNA is composed of two target specifc primers (Primer 1: 5’-CGC ACA GGA ACT GAA GAA TG-3’; Primer 2: 5’-CCG TCG AGA ATA CTG GCA AT-3’).
Each reaction was carried out in a total volume of 5 μL con taining 4.8 μL of qPCR MasterMix Plus as well as 300 nM of each specifc primer, water and 0.2 μL Lambda phage DNA The Lambda DNA standard was serially diluted manually in nuclease free water. The mix was subjected to the following thermal conditions: 95 °C for 3 minutes, followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 25 seconds.
qPCR Assay 2
> Kapa Library quantifcation kit for Illumina® sequencing platforms (Kapa Biosystems, cat # 4824).
Kapa Library quantifcation kit for Illumina sequencing platforms contains the Master Mix, the Primer Mix (Primer 1: 5’-AAT GAT ACG GCG ACC ACC GA-3’; Primer 2: 5’-CAA GCA GAA GAC GGC ATA CGA-3’) and ready-to-use DNA standards. Each reaction was carried out in a total volume of 5 µL containing 4.8 µL of Kapa SYBR Fast qPCR Master Mix containing Primer Premix and water. As template DNA, 0.2 µL DNA standards were used. The mix was subjected to the following thermal conditions: 95 °C for 5 minutes, followed by 35 cycles of 95 °C for 30 seconds and 60 °C for 45 seconds.
Automation
> epMotion 5073l for automated PCR set-up, CleanCap, system incl. Eppendorf MultiCon, epBlue™ software and LH assistant, keyboard, mouse, waste box, 100 – 240 V ±10%/50 – 60 Hz ±5 % (Eppendorf, cat # 5073 000.612)
> TS10 single-channel dispensing tool(Eppendorf, cat # 5280 000.100)
> epT.I.P.S.® Motion 10 µL PCR-clean, with flter, sterile(Eppendorf, cat # 0030 015.193)
> Thermoadapter Frosty (Eppendorf, cat # 5075 789.000)
> Thermoblock for PCR plates, 96-well(Eppendorf, cat # 5075 766.000)
> Eppendorf twin.tec® PCR plates, 96-well, LoBind, semi-skirted (Eppendorf, cat # 5075 129.504)
For each qPCR assay, a method dedicated to the qPCR reaction setup has been programmed. Master Mixes and samples are provided in an Eppendorf twin.tec PCR plates, 96-well, LoBind, semi-skirted. 4.8 µL of the fnal Master Mix is frstly dispensed into the real-time PCR 96-well plates followed by the addition of 0.2 µL DNA template. Negative controls without template were included. Before starting a program, the epMotion surfaces and tools were cleaned using a DNA decontamination solution and treated with UV-light for 15 minutes. The worktable of epMotion 5073l instrument is equipped as on fgure 1.

 

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 reflects 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 buffered solution to ensure stability of the diluted DNA and to reduce DNA adsorption to plastic.
As the physical properties of this buffered solution were different 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 different 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.

Literature

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[3] Gancarek E., Vanbellinghen B., Hamels S. and Art M. Efcient qPCR Setup Without Cross Contamination Using the epMotion Family of Automated Liquid Handling Systems; Eppendorf Application Note 368; 2016368
[4] Desneux J. and Pourcher A. M. Comparison of DNA extraction kits and modifcation of DNA elution procedure for the quantitation of subdominant bacteria from piggery efuents with real-time PCR. MicrobiologyOpen 2014; 3(4): 437–445
[5] Zhang H. and Zhu G. Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro. Front Microbiol. 2015; 6: 991