如何借助epMotion 5073l移液工作站完成微量體積的qPCR反應體系構建
在進行微量體系的液體操作時，如何避免人為誤差與日間變化，如何保證結果的一致性提高實驗效率？如果減少冗長枯燥的重復操作，減少昂貴試劑的使用，提升操作體驗節省實驗經費？
本篇應用旨在介紹如何用10 μL分液工具實現全自動的微量體積的qPCR反應體系構建。在每個PCR反應體系中，樣品量為0.2 μl，單個反應體系總的體積為5 μL。
對于最終的qPCR實驗結果的評估，通過兩個靈敏的qPCR測定來完成。結果顯示借助epMotion 5073l系統可成功地完成微量體積的qPCR體系構建，其精度與重復性都十分出色。
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 di?erent 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 o?ers 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 e?ect on the correct handling of small volumes.
With so many variables a?ecting 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.