如何借助epMotion 5073l移液工作站完成微量体积的...（一）

如何借助epMotion 5073l移液工作站完成微量体积的qPCR反应体系构建

在进行微量体系的液体操作时，如何避免人为误差与日间变化，如何保证结果的一致性提高实验效率？如果减少冗长枯燥的重复操作，减少昂贵试剂的使用，提升操作体验节省实验经费？

本篇应用旨在介绍如何用10 μL分液工具实现全自动的微量体积的qPCR反应体系构建。在每个PCR反应体系中，样品量为0.2 μl，单个反应体系总的体积为5 μL。

对于最终的qPCR实验结果的评估，通过两个灵敏的qPCR测定来完成。结果显示借助epMotion 5073l系统可成功地完成微量体积的qPCR体系构建，其精度与重复性都十分出色。

he 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.