LightCycler® 1536 Application Note No. 1

Introduction

Miniaturizing quantitative PCR (qPCR) using 1536-multiwell plates results in increased throughput, reagent savings, and error reduction. However, delivering reagents to high-density plates with 1,536 wells is challenging using conventional tip-based liquid handling systems. Problems include inaccurate pipetting and cross-contamination between wells in the multiwell plate.

Echo® 500 Series Liquid Handlers from Labcyte® use neither tips nor nozzles. The dispensing mechanism never touches the reagents in the wells, thereby significantly eliminating the risk of well-to-well cross-contamination.

The LightCycler® 1536 Real-Time PCR Instrument is thehigh-throughput member of the widely used LightCycler®Real-Time PCR family.

In the present study, we performed three experiments toinvestigate how the Echo® 555 Liquid Handler functionswith the LightCycler® 1536 System in miniaturized qPCR with high precision and accuracy. A special focus wasgiven to testing for cross-contamination.

For life science research only. Not for use in diagnostic procedures.

January 2012

Celeste Glazer, Maria Sonntag, Carl Jarman, Sammy Datwani *Gregor Sagner, Armin Tgetgel, Gudrun Tellmann+

* Labcyte, Inc., Sunnyvale, CA, USACorresponding author: celeste.glazer@labcyte.com

+ Roche Applied Science, GermanyCorresponding author: gudrun.tellmann@roche.com

Miniaturized Quantitative PCR using the Echo® 555 Liquid Handler and the LightCycler® 1536 Real-Time PCR System

Miniaturized Quantitative PCR using the Echo® 555 Liquid Handler and the LightCycler® 1536 Real-Time PCR System22

Experiment 1: Echo® Liquid Handler Dispensing Precision and Accuracy

To measure transfer volume performance of the Echo® Liquid Handler for PCR reagents, dispensing performance was measured using dual-dye ratiometric photometry.

Dispensing precision and accuracy were tested using the Artel MVS® (Multi-Channel Verification System; www.artel-usa.com). Solutions of premixed primers and probes were combined with MVS dyes. Aliquots of 10 µl volumes were loaded into a 384LDV (low-dead-volume) Echo® Qualified Source Plate.

Subsequently, 0.1, 0.5 and 1.0 µl volumes were transferred from the source plate to each well of an empty (dry) 384-well destination plate (Artel) using the Echo® 555 Liquid Handler. Finally, 55 µl of diluent were added. The plate was centrifuged, incubated at +15 to +25°C for 30 minutes, and an absorbance measurement was taken.

Table 1 shows the obtained volume, relative accuracy, standard deviation, and coefficient of variation (CV) for each target volume. Transfer volume accuracy and precision were excellent, with accuracy above 96% and precision below 4% CV.

Echo® 555 Liquid Handler Volumetric Precision and Accuracy

Target volume (µl) 0.100 0.500 1.000

Mean volume for all wells (µl) 0.104 0.511 1.036

Relative accuracy for all wells 96.0% 97.8% 96.4%

Standard deviation for all wells (µl) (SD) 0.004 0.011 0.025

Coefficient of variation (CV) for all wells 3.46% 2.15% 2.41%

Table 1. Echo® 555 Liquid Handler Performance

Materials, Methods and Results

Echo® 555 Liquid Handler

The Labcyte® Echo 500 series revolutionizes liquid transfer using acoustic energy to eject fluids. The instrument focuses sound waves directly under a microplate well, and ‘listens’ for the reflection (or echo) to determine the composition and fluid height. This happens automatically – in milliseconds – allowing for precise and accurate transfer of 2.5 nl droplets into an inverted microplate positioned above. Large volume transfer is achieved by transferring several hundred droplets per second.

Traditional liquid handlers have a two-step aspirate and dispense process with an optimal volume range for accurate and precise transfers. Echo liquid handlers transfer 2.5 nl repeatedly, so precision and accuracy are consistent over a larger volume range. Echo® precision for low nanoliter transfers is unmatched by tip-based, motor-driven liquid handlers.

For further details regarding the Echo 550 liquid handler, visit: www.labcyte.com

Echo® 550 Liquid Handler specifications:

Dimensions W 53.9 cm x D 68.3 cm x H 92.5 cm

Weight 128 kg

Drop Transfer Volume

2.5 nl

Volume Transfer Range

2.5 nl to 10,000 nl

Transfer Accuracy < 10% deviation from target volume

Transfer Precision < 8% CV

Destination Labware:

All Echo® Qualified Microplates and most ANSI-compliant/SBS-standard microplates in 96-, 384-, 1536-, and 3456-well formats, 16 mm in height

Application Software (optional):

Echo® Software applications: Echo® Plate Reformat, Echo® Dose-Response, Echo® Cherry Pick

555 Liquid Handler

The Labcyte® Echo 500 series revolutionizes liquid transfer using acoustic

Miniaturized Quantitative PCR using the Echo® 555 Liquid Handler and the LightCycler® 1536 Real-Time PCR System 33

LightCycler® 1536 Real-Time PCR SystemHigh Throughput – Redefined

The LightCycler® 1536 System, a member of the LightCycler® Real-Time PCR family, embodies high throughput, using an ultrafast instrument, intuitive software, and the unique LightCycler® 1536 Multiwell Plates with next generation real-time PCR reagents. Precision technology has produced high performance optics combined with a modification of the block cycler used in the proven LightCycler® 480 Instruments for thermal accuracy combined with speed, versatility and reliability.

LightCycler® 1536 Software seamlessly integrates the LightCycler® 1536 Instrument into the PCR workflow. The software performs basic real-time PCR calculations. It also contains an automation integration module (AIM) that allows for full connectivity, to both remotely control the LightCycler® 1536 System and to exchange, manage and easily allocate experimental data with other software in network and LIMS environments. A Software Development Kit can be deployed by 3rd party automation vendors or end users to establish remote applications between the LightCycler® 1536 System and other instrumentation.

The LightCycler® 1536 Multiwell Plate features Thermaxis® technology. An insulating top layer is incorporated with the thermally conductive wells, precisely molded to fit the distinctive V-groove of the instrument’s thermal block. This revolutionary plate design enables small reaction volumes of just 0.5 to 2.0 µl – without compromising sensitivity or reproducibility.

1536 Real-Time PCR SystemHigh Throughput – Redefined

Figure 1. LightCycler® 1536 Multiwell Plates (front and back view).

Figure 2. Schematic of the LightCycler® 1536 Multiwell Plate.

Thermal block cycler cover

Heat-insulating layer

Heat-conducting layer (PCR well)

Thermal block cycler mount

Miniaturized Quantitative PCR using the Echo® 555 Liquid Handler and the LightCycler® 1536 Real-Time PCR System4

Materials, Methods and Results continued

The LightCycler® 1536 System uses function tested LightCycler® 1536 DNA Master Reagents designed for the most demanding applications in automated high-throughput PCR workflows. Featuring innovations, such as (i) 5x concentrated reagents for low-volume PCR reactions, (ii) exceptional room temperature stability of at least 48 hours, and (iii) novel error-tracking systems, these reagents are the next generation in real-time PCR chemistry.

For further details regarding the LightCycler® 1536 System, visit: www.lightcycler1536.com

LightCycler® 1536 System Specifications

Dimensions W 57.4 cm x D 58.8 cm x H 49.7 cm

Weight 55.6 kg

Reaction volume 0.5–2.0 µl

Run time < 50 min

Temperature Range +37 to +95°C

Heating Rate 4.8°C/sec

Cooling Rate 2.5°C/sec

Temperature Control • Peltier-based heating/cooling • Therma-BaseTM heat spreading technology

Excitation Xenon Lamp

Detector Cooled monochrome CCD camera

Filters (selectable) Excitation (nm): 465, or 533Detection (nm): 510, or 580

Multiwell Plate • Thermaxis® technology (two-component design)• Barcode labeled for automated workflow tracking

Software • User-friendly programming • Compact overview of results (e.g., crossing points, slopes, endpoint fluorescence) and error tracking surveillance information• Novel threshold functions for data refinement • Powerful tools for data sorting and reviewing • Multiple data input/output formats• Simple data management for network environments• Result data accessible via LIMS

Fast high-content gene expression analysis is made possible using Roche’s RealTime ready assays using Universal ProbeLibrary probes, and RealTime ready DNA Probes Master reagents that eliminate time-consuming assay optimization.

Miniaturized Quantitative PCR using the Echo® 555 Liquid Handler and the LightCycler® 1536 Real-Time PCR System 5

Experiment 2: PCR Volume Miniaturization using the LightCycler® Instrument and Echo® 555 Liquid Handler

To evaluate miniaturization of the PCR volume in the LightCycler® 1536 Instrument, 0.5 µl and 1.0 µl transfer volumes were tested for dispensing by the Echo® 555 Liquid Handler.

Each PCR reaction contained the following: 500 pg/µl of cDNA, 500 nM GAPDH primer, 400 nM UPL Probe #60, and 1x RealTime ready DNA Probes Master. The reaction mix was added to 96 wells of a 384-well polypropylene Echo® Qualified Source Plate. The Echo® 555 Liquid Handler was used to add 0.50 and 1.0 µl, respectively, of the reaction mix across one entire LightCycler® 1536 Multiwell Plate. Afterwards both plates were sealed using the Agilent PlateLoc heat sealer with Clear Weld Seal Mark II (4titude® Ltd.). PCR was performed as follows: Pre-denaturation step: +95°C for 60 seconds, followed by PCR amplification: +95°C for 0 seconds, +60°C for 60 seconds, for 45 cycles. After the run, PCR results were analyzed using LightCycler® 1536 Software.

As shown in Table 2, results for both, 0.5 µl and 1.0 µl, show low standard deviations and good coefficients of variation (CVs). The average CV for the Cp values was consistent at approximately 1%. Workflow robustness and reproducibility are shown by the distinct, homogeneous profile of the amplification curves (see Figure 3).

Total plate volume 0.5 µl 1.0 µl

Average crossing point value (Cp) 22.38 22.03

Cp standard deviation (SD) 0.21 0.21

Coefficient of variation (CV) 0.94% 0.97%

Cp minimum 21.61 20.98

Cp maximum 23.09 22.53

Cp range 1.48 1.54

Table 2. Echo® Liquid Handler Volume Testing using Real-Time PCR

Figure 3. Amplification curves of a 1.0 µl qPCR experiment in the LightCycler® 1536 Instrument, showing high crossing point (Cp) reproducibility. Results of the same quality were also obtained with the 0.5 µl setting (see Table 2).

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65

/510

Cycles

1.000.950.900.850.800.750.700.650.600.550.500.450.400.350.300.250.200.150.100.050.00

5 10 15 20 25 30 35 40 45

Miniaturized Quantitative PCR using the Echo® 555 Liquid Handler and the LightCycler® 1536 Real-Time PCR System6

Figure 5. LightCycler® 1536 Software Results: (a) Heatmap display using the setting Call. The call option of heatmap analysis mode provides the calculated value of a real-time PCR sample in a given well. The color green shows a positive PCR reaction, and red indicates a negative PCR reaction in a well; (b) This panel shows the corresponding LightCycler® 1536 amplification curves. As shown in Figure 5 using the heatmap mode of the LightCycler® 1536 Software, there was no evidence of cross-contamination in the combined workflow using the Echo® 555 Liquid Handler and LightCycler® 1536 Instrument.

Materials, Methods and Results continued

Figure 4. Plate setup for the cross-contamination test

Quadrant 1 Quadrant 2

Quadrant 3 Quadrant 4

       

A

Experiment 3: No Evidence of Cross-Contamination using the LightCycler® 1536 System and Echo® 555 Liquid Handler

Cross-contamination was evaluated using a checkerboard pattern, alternating the position of the positive control in each quadrant of a LightCycler® 1536 Multiwell Plate (see Figure 4). Positive samples (�) and negative samples (�) were transferred from a 384-well polypropylene Echo qualified source plate into a LightCycler® 1536 Multiwell Plate using the Echo® 555 Liquid Handler. This experiment was done to show that positive wells would not contaminate the eight surrounding negative sample wells, independent of dispensing order or relative positions in the grid. Each quadrant was duplicated sixteen times in each corner of the 1,536-well destination plate.

For this cross-contamination test, reaction volumes of 0.5 µl were transferred from a 384-well polypropylene plate to the LightCycler® 1536 Multiwell Plate. Positive samples consisted of 500 nM HPRT primer, 400 nM HPRT UPL Probe, 1x RealTime ready DNA Probes Master, and 500 pg cDNA (qPCR Human Reference cDNA, random primed, Clontech). Negative samples consisted of 500 nM primers, 300 nM probe, and 1x RealTime ready DNA Probes Master. Real-time PCR was performed using the LightCycler® 1536 Instrument as follows: pre-denaturation step: +95°C for 60 seconds; amplification: +95°C for 0 seconds, +60°C for 60 seconds for 45 cycles. After the run, PCR results were analyzed using LightCycler® 1536 Software.

1.10

1.00

0.90

0.80

0.70

0.60

0.50

0.40

0.30

0.20

0.10

0.05

0.00

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33

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Cycles 5 10 15 20 25 30 35 40 45

B

Miniaturized Quantitative PCR using the Echo® 555 Liquid Handler and the LightCycler® 1536 Real-Time PCR System 7

The combination of the the Echo® 555 liquid handler and LightCycler® 1536 Instrument is a good solution for high-throughput automated workflows, achieving reproducible precise real-time PCR, gene expression profiling (see Figures 4 and 5), and endpoint genotyping (data not shown).

Assay miniaturization produced accurate and precise results using the appropriate liquid transfer instrument. The Echo® 555 Liquid Handler supports reproducible, high-throughput quantitative analyses with the LightCycler® 1536 Instrument, using as little as 0.5 µl volumes. Echo® Liquid Handler dispensing precision and accuracy was shown by the very low obtained Cp standard deviation and coefficients of variation. The Echo® 555 Liquid Handler accurately transfers 0.5 µl PCR reagent volumes from a 384-well plate into LightCycler® 1536 Multiwell Plates without cross-contamination.

Conclusions

The synergy of Echo® 555 Liquid Handler and LightCycler® 1536 System technologies conserves reagents, tips and washing steps during plate setup. This is achieved by the tipless technology of the Echo® Liquid Handler, and the ultralow volume plate-based LightCycler® 1536 Real-Time PCR Instrument. This workflow excels in generating high quality data in a short period of time using minimal reagent quantities.

Ordering Information

Product Cat. No. Pack Size

LightCycler® 1536 Instrument 05 334 276 001 1 Instrument

LightCycler® 1536 Software 1.1 06 569 382 001 1 Software Package

LightCycler® 1536 Multiwell Plate 05 358 639 001 100 Plates

RealTime ready DNA Probes Master (5x concentrated)

05 502 381 001 5 x 1 ml (12,500 x 2 µl reactions, or 1,250 x 20 µl reactions)

LightCycler® 1536 DNA Green Master (5x concentrated)

05 573 092 001 5 x 1 ml (12,500 x 2 µl reactions, or 1,250 x 20 µl reactions)

For detailed information, please visit: www.lightcycler1536.com

RealTime ready Assays For detailed information, please visit: www.realtimeready.roche.com

Universal ProbeLibrary Assays For detailed information, please visit: www.universalprobelibrary.com

Echo® 550 Liquid Handler 550-001-FCP-O 1 Instrument

Echo® Plate Reformat Software ECHO-Reformat 1 Software Package

Echo® Qualified 384-Well Polypropylene Microplate P-05525 100 Plates

Echo® Qualified 384-Well COC Microplate, Low Dead Volume

LP-0200 100 Plates

For detailed information, please visit: www.labcyte.com

Published byRoche Diagnostics GmbHSandhofer Straße 11668305 MannheimGermany

© 2012 Roche Diagnostics.All rights reserved.

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For life science research only. Not for use in diagnostic procedures.

LIGHTCYCLER and REALTIME READY are trademarks of Roche. Thermaxis is a trademark of IT-IS International Ltd.

Echo und Labycyte are trademarks of Labcyte Inc.The LightCycler® 1536 Multiwell Plate is manufactured under license from IT-IS International Ltd.

Exiqon and ProbeLibrary are registered trademarks of Exiqon A/S, Vedbaek, Denmark.

All other product names and trademarks are the property of their respective owners.