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Application Note G103 | Echo Liquid Handler, Access Workstation

Automated RT-qPCR Utilizing the Access™ Workstation, the Echo® Liquid Handler, and RealTime Ready™ Reagents from Roche Applied Science

Celeste Glazer, Randy Dyer Labcyte Inc.

AbstractRT-qPCR has been increasingly utilized by researchers to quantify mRNA levels. Its widespread

use has been limited by an often laborious multi-step process and high reagent cost. Recent

advances in non-contact reagent dispensing and next generation one step lysis reagents are

enabling scientists to generate high throughput data at a much faster rate. This technical note

describes the automation of three complementary tools to generate high throughout RT-qPCR

data; the Echo liquid handler, RealTime Ready cell lysis reagents, and the Access workstation.

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Application Note G103

IntroductionWith a high level of precision, specificity, and sensitivity, reverse transcription quantita-tive real-time PCR (RT-qPCR) is the preferred method for quantitative gene expression. In drug development laboratories, RT-qPCR analysis to verify cellular mRNA expression levels has traditionally followed reporter-gene assays for follow-up confirmation. Higher reagent costs and the requirement for intermediate purification have hindered the use of RT-qPCR as a primary screening tool.

A completely automated workflow has been developed at Labcyte, pairing three sig-nificant developments; the Echo liquid handler, the Access workstation, and one step reagent chemistries create a hands free workflow that utilizes low volume reactions to save reagents and time. The Echo liquid handler transfers nanoliter volumes of reagents using acoustic energy. Since the Echo liquid handler has no tips or nozzles it removes the cost of tips and the risk of clogging and cross-contamination from the workflow. The Access workstation integrates the Echo liquid handler with complementary instruments in a compact footprint. Tissue-culture treated microplates (384-well and 1536-well) are also utilized for consistent acoustic transfer of lysates from a small format. By pairing next generation reagent chemistries that require fewer steps with the highly accurate and precise low volume liquid handling capabilities of the Echo liquid handler, Labcyte has developed a workflow and set of procedures that enable miniaturized gene expression profiling with RT-qPCR.

The Echo liquid handler transfers nanoliter volumes of reagents using acoustic energy.

Echo Liquid Handler | Access Workstation

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One-step qPCR preparationRecent innovations in RT-qPCR reagent kits have removed the need for time-consuming, multi-step mRNA purification and cDNA synthesis procedures in what is referred to as a “one-step” workflow. In gene expression studies, cell lines are screened against compounds or siRNA. In a conventional process, cells are lysed and mRNA is purified. Complementary DNA (cDNA) is produced by reverse transcription and purified. The cDNA is then used as a template for RT-qPCR.

In a “one-step” RT-qPCR process, treated cells are lysed. The lysed mixture is then added to the PCR reagent, which incorporates two enzymes: one to perform the reverse transcription, and another to perform the cDNA amplification. This process significantly reduces hands-on time, simplifies reagent transfers, and reduces overall time for gene expression data generation.

One Step Gene Expression

Real-time PCR

RealTime Ready Cell Lysis

Transcriptor Universal cDNA

Master

RealTime Ready Cell Lysis

Real-time PCR

Column Based RNA Isolation

cDNA Synthesis Real-time PCR

Conv

enie

nce

Time-to-Result (minutes)30 60 90 120 150

1-Step Accelerated Workflow

2-Step Accelerated Workflow

Conventional Workflow

Figure 1 This diagram illustrates the improvement in time to results that can be expected when implementing RealTime Ready Cell Lysis

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Application Note G103

To validate the miniaturization of this process using the Echo liquid handler, lysates and reagents were transferred in a “one-step” workflow into qPCR microplates and analyzed using the LightCycler 1536 system from Roche.

Cells were plated into Echo® qualified tissue culture treated plates, lysed and transferred with RNA Probes Master solution into a 1536 qPCR microplate. Using the Echo liquid handler this “one-step” workflow can reduce the total qPCR volumes to less than one microliter.

Materials• Human fibrosarcoma cell line HT1080 (Sigma, 85111505-1VL)• DMEM, High Glucose, no Glutamine, no Phenol Red (Life Technologies, 31053-036)• Fetal Bovine Serum (Life Technologies, 10439-024)• MEM Non-essential Amino Acid solution (Life Technologies, 1114-0050)• L-Glutamine-200 mM (Life Technologies, 25030-081)• 0.25% Trypsin-EDTA with phenol red (Life Technologies, 25200-056)• Penicillin/Streptomycin (Life Technologies, 15140-122)• Phosphate buffered saline (PBS) pH 7.4• RealTime Ready Cell Lysis Kit (Roche Applied Science, 05 943 523 001)• RNA Probes Master (Roche Applied Science, 04 991 885 001) • PCR grade water• Universal ProbeLibrary Reference Gene Assays (Roche Applied Science)

Human G6PD Gene Assay (05 046 246 001)Human GAPD Gene Assay (05 190 541 001)Human PBGD Gene Assay (05 046 157 001)

• Echo qualified 384-well (PPT-05525) or 1536-well (LPT-0400) tissue culture treated microplates

• Echo qualified 384-well (LP-0200) or 1536-well low dead volume (LP-0400) microplates• Tissue culture incubator (37°C, 5% CO2, 95% humidity)• LightCycler 1536 multiwell qPCR plate (Roche Applied Science 05 358 639 001)• LightCycler 1536 Instrument (Roche Applied Science 05 334 276 001)• Agilent PlateLoc thermal microplate sealer (Agilent G5402A)• Clear permanent seal (Agilent 24212-001)

Figure 2 This diagram provides an over-view of a “one-step” procedure using the RealTime Ready Cell Lysis Kit from Roche utilizing the Access workstation.

Echo Liquid Handler | Access Workstation

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Labcyte Access™ workstation configuration:

a. Labcyte Echo 555 or Echo 550 liquid handlerb. Thermo Labsystems Multidrop Combi reagent dispenserc. BioTek® EL406 microplate washer/dispenserd. Agilent PlateLoc thermal microplate sealere. Agilent Microplate Centrifugef. Four Labcyte 20-plate random access racksg. Keyence barcode readerh. Labcyte four shelf lid removal stationi. Tempo™ automation control software

Top Down View of Access Workstation

LightCycler

RacksSealer

Echo 555

Walk up view of Access Workstation

RacksWasher

The Access workstation can be configured to meet a range of workflow requirements.

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Application Note G103

Methods and ResultsCell plate preparation Lidded 384-well Echo qualified tissue culture microplates were placed in rack 1 of the Access workstation. A single Tempo software protocol was initiated to coordinate dis-pensing of cells and sample transfer on the Access workstation. The Access plate han-dling robot transferred each tissue culture microplate to the Multidrop Combi dispenser for cell plating. Tissue culture microplate lids were automatically removed before place-ment at the dispenser and replaced after dispensing was complete. HT1080 cells grown in flasks to a concentration of 220,000-550,000 cells/mL were dispensed by the Multidrop Combi into every well of each microplate providing approximately 2000-5000 cells per well. Subsequently, each tissue culture microplate was re-lidded, centrifuged (1500 x g), and returned to rack 1 of the Access system. Cell microplates were transferred to an incubator and incubated overnight at 37°C, 5% CO2, 95% humidity. Scientists wishing to treat cells with an agonist would incorporate the Echo liquid handler into the Tempo protocol to dispense the compound.

qPCR preparationqPCR master mix was prepared offline following manufacturer’s instructions using the following ratios: to prepare the mix for more than one reaction, multiply the amount in the “Volume” column by the number of reactions to be run + one additional reaction.

Component Conc. Volume (uL) Final Conc.

LightCycler® 480 RNA Master Hydrolysis Probes

2.7X 0.37 1.0X

Activator 50 mM 0.07 3.35 mM

Enhancer 20X 0.05 1.0X

Thermolabile nuclease 40X 0.03 1U

UPL Primer Set 20uM 0.02 400 nM

UPL Probe 10uM 0.03 300 nM

Water, PCR grade 0.34

Total Volume Master Mix 0.90

Lysate Addition 0.10

Total Reaction Volume 1.00

Sufficient master mix was prepared in advance to address an entire batch of 1536-well qPCR plates. In an Echo qualified 384-well polypropylene source microplate, 16 wells containing 65 uL of master mix were used to transfer 450 nL of master mix to each well of the 1536-well qPCR plate. Cell lysate prepared in step two accounted for 10% of the desired final assay volume (50 nL).

HT1080 cells were plated into 384-well tissue culture microplates and incubated overnight prior to lysis.

Echo Liquid Handler | Access Workstation

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Cell lysis and qPCR assay additionA Tempo software protocol, controling the Access workstation, coordinated the wash, addition of lysis buffer, incubation of the lysed cells, and addition of the master mix to the qPCR microplate. The cell plates in rack 1 were de-lidded and transferred to the BioTek EL406™ washer. Wells were washed twice with 50 uL of PBS. After the wash step a secondary manifold on the EL406 washer dispensed 20 uL of RealTime Ready Cell Lysis buffer to each well. Each cell plate was re-lidded and returned to rack 1 to incubate at room temperature for five minutes. The process was run sequentially so each plate maintained the same incubation time.

As the cell plates were being washed, the robot loaded the master mix source plate and qPCR destination plate into the Echo liquid handler. The Echo Plate Reformat protocol used the fixed plate feature to dispense the master mix to each destination qPCR plate. After addition of the master mix, each destination qPCR plate was placed back on the rack in preparation for the addition of lysate from the cell microplate. From rack 1, a cell plate containing lysate was de-lidded and transferred to the centrifuge and spun at 1500 x g for two minutes then transferred to the source stage of the Echo liquid handler where 50 nL of cell lysate was transferred into each well of the 1536-well qPCR plate. After lysate transfer the cell plate was re-lidded and returned to rack 1. The 1536-well qPCR plate was sealed with the PlateLoc thermal microplate sealer (160°C, 2 seconds), followed by a centrifugation (1500 x g, 1 minute) before returning to rack 4. See diagram below for location of plate in device with timing.

qPCROnce all qPCR plates were prepared they were processed on the LightCycler 1536 sys-tem. This system can be integrated with the Access workstation for automated loading. The LightCycler 1536 System was loaded and a protocol was initiated which included a pre-incubation at 60°C for 10 minutes, followed by 95°C denaturation. The plate was then cycled for 45 cycles with an initial 95°C 10 sec denaturation, and subsequent 63°C annealing time, with a final cool down of 40°C for 2 minutes.

Figure 3 Tempo automation control software coordinates all of the plate handling steps on the Access workstation

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5. Instrument protocols and parametersDispense order utilizing the Echo Plate Reformat SoftwareAssays were stamped across a 1536 qPCR plate using the “fixed” plate setting. Three ref-erence gene assays (GAPDH, G6PD, and PBGD) were used as representative low, medium and high copy reference genes (shown as green, yellow and blue colors below).

Each source plate was then transferred into two quadrants of the qPCR plate.

Echo Plate Reformat soft-ware enables users to easily set up protocols for any well to any well transfers.

Echo Liquid Handler | Access Workstation

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Tempo Software ProtocolTempo software allows the user to map their source and destination plates in relation to the Echo protocol.

Pre and post run actions were selected for each set of source and destination plates. Pre- run actions include: washing source plates, dispensing lysis buffer and centrifugation. Post-run actions include sealing the plates and centrifugation of the destination plates prior to placement in the LightCycler 1536 system.

Tempo software wizards guide the user plate map-ping and pre and post run actions.

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6. ResultsThree genes on each 384-well microplate were analyzed based on expected copy number (high, medium, and low copy). Three 384-well plates were transferred in duplicate into three 1536-well qPCR destination plates to generate six replicates for each gene.

Plate 1

GAPDH G6PD PBGD

Min 20.96 21.06 26.53 26.93 29.51 29.55

Max 23.89 24.17 29.84 29.82 33.82 34.45

Average 22.49 22.69 28.09 28.40 31.10 31.47

St. Dev 0.64 0.72 0.68 0.64 0.93 0.91

CV 2.82% 3.17% 2.41% 2.25% 3.00% 2.88%

Plate 2

GAPDH G6PD PBGD

Min 21.38 21.85 27.14 27.80 28.26 29.44

Max 25.79 26.07 30.50 30.56 32.86 33.47

Average 22.73 23.08 28.51 29.00 30.89 31.46

St. Dev 0.77 0.63 0.64 0.58 0.74 0.66

CV 3.37% 2.71% 2.25% 1.99% 2.39% 2.09%

Plate 3

GAPDH G6PD PBGD

Min 20.17 19.43 25.73 27.15 28.08 28.86

Max 24.27 23.72 30.36 30.19 32.28 32.83

Average 22.38 22.00 28.66 28.78 30.42 30.88

St. Dev 0.78 0.93 0.80 0.70 0.77 0.71

CV 3.49% 4.21% 2.79% 2.42% 2.52% 2.29%

Plate to Plate Comparison

GAPDH G6PD PBGD

Plate 1 22.49 22.69 28.09 28.40 31.10 31.47

Plate 2 22.73 23.08 28.51 29.00 30.89 31.46

Plate 3 22.38 22.00 28.66 28.78 30.42 30.88

Low, medium and high Cp values correspond with expected starting copy number.

Echo Liquid Handler | Access Workstation

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Results show a consistent and repeatable result for 384 samples dispensed in duplicate to three 1536 qPCR plates with reference genes ranging from an average of 22 Cp to an average of 30 Cp. Standard deviations for the three plates range from 0.58 to 0.93 with CVs varying from 2-4%. Time to completion for these three, 384 well source plates run on the Access workstation stamped into three, 1536-well qPCR destination plates was 1 hr and 10 minutes.

ConclusionsThe use of RT-qPCR as a primary high throughput method to accurately quantify gene expression is commonly perceived to be inefficient. The cost of reagents, consumed sample, and tedious preparation methods required for RT-qPCR has lead researchers to rely on alternative methods with lower performance, reserving RT-qPCR for follow-up confirmation only.

As outlined above, using the Access workstation in combination with a “one-step” qPCR preparation workflow, RT-qPCR can be adapted for high throughput gene expression screening. The ability of the Echo liquid handler to transfer nanoliter volumes of sample and reagents with incomparable accuracy and precision reduces screening costs to make one-step RT-qPCR a viable primary screening method.

By comparison to traditional alternatives, one-step high throughput RT-qPCR is efficient, economical, and broadly applicable. With the Access workstation, RT-qPCR is easily implemented as a high throughput primary screening method capable of producing tens of thousands of data points per day.

Figure 4 Results show consistent Cp values for all three genes of interest across multiple plate runs

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