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

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1 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. Abstract RT-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. labcyte.com 1

2 Application Note G103 Introduction With a high level of precision, specificity, and sensitivity, reverse transcription quantitative 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. The Echo liquid handler transfers nanoliter volumes of reagents using acoustic energy. A completely automated workflow has been developed at Labcyte, pairing three significant 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. 2 labcyte.com

3 Echo Liquid Handler Access Workstation One-step qpcr preparation Recent 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 1-Step Accelerated Workflow Figure 1 This diagram illustrates the improvement in time to results that can be expected when implementing RealTime Ready Cell Lysis Convenience Real-time PCR RealTime Ready Cell Lysis 2-Step Accelerated Workflow Transcriptor Universal cdna Master Real-time PCR RealTime Ready Cell Lysis Conventional Workflow Column Based RNA Isolation cdna Synthesis Real-time PCR Time-to-Result (minutes) labcyte.com 3

4 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. Figure 2 This diagram provides an overview of a one-step procedure using the RealTime Ready Cell Lysis Kit from Roche utilizing the Access workstation. 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, VL) DMEM, High Glucose, no Glutamine, no Phenol Red (Life Technologies, ) Fetal Bovine Serum (Life Technologies, ) MEM Non-essential Amino Acid solution (Life Technologies, ) L-Glutamine-200 mm (Life Technologies, ) 0.25% Trypsin-EDTA with phenol red (Life Technologies, ) Penicillin/Streptomycin (Life Technologies, ) Phosphate buffered saline (PBS) ph 7.4 RealTime Ready Cell Lysis Kit (Roche Applied Science, ) RNA Probes Master (Roche Applied Science, ) PCR grade water Universal ProbeLibrary Reference Gene Assays (Roche Applied Science) Human G6PD Gene Assay ( ) Human GAPD Gene Assay ( ) Human PBGD Gene Assay ( ) 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% CO 2, 95% humidity) LightCycler 1536 multiwell qpcr plate (Roche Applied Science ) LightCycler 1536 Instrument (Roche Applied Science ) Agilent PlateLoc thermal microplate sealer (Agilent G5402A) Clear permanent seal (Agilent ) 4 labcyte.com

5 Echo Liquid Handler Access Workstation Top Down View of Access Workstation The Access workstation can be configured to meet a range of workflow requirements. LightCycler Echo 555 Labcyte Access workstation configuration: Sealer Racks Walk up view of Access Workstation Washer Racks a. Labcyte Echo 555 or Echo 550 liquid handler b. Thermo Labsystems Multidrop Combi reagent dispenser c. BioTek EL406 microplate washer/dispenser d. Agilent PlateLoc thermal microplate sealer e. Agilent Microplate Centrifuge f. Four Labcyte 20-plate random access racks g. Keyence barcode reader h. Labcyte four shelf lid removal station i. Tempo automation control software labcyte.com 5

6 Application Note G103 HT1080 cells were plated into 384-well tissue culture microplates and incubated overnight prior to lysis. Methods and Results Cell 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 dispensing of cells and sample transfer on the Access workstation. The Access plate handling robot transferred each tissue culture microplate to the Multidrop Combi dispenser for cell plating. Tissue culture microplate lids were automatically removed before placement at the dispenser and replaced after dispensing was complete. HT1080 cells grown in flasks to a concentration of 220, ,000 cells/ml were dispensed by the Multidrop Combi into every well of each microplate providing approximately 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% CO 2, 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 preparation qpcr 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 X Activator 50 mm mm Enhancer 20X X Thermolabile nuclease 40X U UPL Primer Set 20uM nm UPL Probe 10uM 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). 6 labcyte.com

7 Echo Liquid Handler Access Workstation Cell lysis and qpcr assay addition A 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. Figure 3 Tempo automation control software coordinates all of the plate handling steps on the Access workstation qpcr Once all qpcr plates were prepared they were processed on the LightCycler 1536 system. 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. labcyte.com 7

8 Application Note G Instrument protocols and parameters Dispense order utilizing the Echo Plate Reformat Software Assays were stamped across a 1536 qpcr plate using the fixed plate setting. Three reference 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). Echo Plate Reformat software enables users to easily set up protocols for any well to any well transfers. Each source plate was then transferred into two quadrants of the qpcr plate. 8 labcyte.com

9 Echo Liquid Handler Access Workstation Tempo Software Protocol Tempo software allows the user to map their source and destination plates in relation to the Echo protocol. Tempo software wizards guide the user plate mapping and pre and post run actions. Pre and post run actions were selected for each set of source and destination plates. Prerun 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. labcyte.com 9

10 Application Note G Results Three 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 Low, medium and high Cp values correspond with expected starting copy number. Max Average St. Dev CV 2.82% 3.17% 2.41% 2.25% 3.00% 2.88% Plate 2 GAPDH G6PD PBGD Min Max Average St. Dev CV 3.37% 2.71% 2.25% 1.99% 2.39% 2.09% Plate 3 GAPDH G6PD PBGD Min Max Average St. Dev CV 3.49% 4.21% 2.79% 2.42% 2.52% 2.29% Plate to Plate Comparison GAPDH G6PD PBGD Plate Plate Plate labcyte.com

11 Echo Liquid Handler Access Workstation Figure 4 Results show consistent Cp values for all three genes of interest across multiple plate runs 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. Conclusions The 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. labcyte.com 11

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