For general laboratory use. DEVELOP YOUR LAB, YOUR WAY€¦ · · 2015-10-06instrument and assay concept allows you to truly develop your lab, your way. FLOW Classic ... Liquid

DEVELOP YOUR LAB, YOUR WAYThe FLOW Solution: Expand your potential today

Technical brochure

www.flow.roche.com

Published by Roche Molecular Systems, Inc. 4300 Hacienda Drive Pleasanton, CA 94588 USA

© 2015 Roche Molecular Systems Inc.

The FLOW Solution is not available in all territories due to different national regulations.This document is not intended for use in the USA. For general laboratory use.

LIGHTCYCLER and MAGNA PURE are registered trademarks of Roche.All other product names and trademarks are the property of their respective owners.

Redesign your lab with the FLOW Solution

The FLOW Solution is a highly flexible modular, semi-automated data and sample workflow. It seamlessly processes various sample and tube types in a completely traceable barcoded manner from primary sample handling, NA extraction and PCR setup to qPCR. LIMS connectivity, middleware software management and a clever IT structure enables a smooth data transition without leaving room for manual errors. The modular instrument and assay concept allows you to truly develop your lab, your way.

FLOW Classic

FLOW FlexFLOW Flex is the minimal instrument configuration necessary to be supported by the FLOW Software. It consists of a PCR Setup Instrument, a MagNA Pure 96 and a Roche qPCR Instrument. FLOW Flex operates with one pipetting robot used for both Primary Sample Handling and PCR Setup. It generates a non linear sample workflow with dual use of the Roche Liquid Handling Platform (PCR Setup Instrument).

FLOW Classic is the same instrumentation configuration as FLOW Flex with an additional pipetting robot. This could be either a Primary Sample Handling Instrument or a PCR Setup Instrument. In this configuration, the workflow is linear and has dedicated tasks for each pipetting robot.

Expand your possibilities

There are two ways to grow your FLOW Solution: Install an additional FLOW Flex or FLOW Classic or add extraction and amplification systems to an existing workflow. Here are some examples for options:

FLOW Ultra

FLOW FusionFLOW Fusion is a software application that allows you to run two separate FLOW installations in a coordinated manner. It allows for independent loading of samples and generates more flexible workflow options. This software tool operates both, linear and nonlinear workflows. To support this configuration a FLOW Software Additional License is necessary. FLOW Fusion and FLOW Software Additional License will be available at the launch of FLOW Software version 2.1.

FLOW Ultra is a configuration which expands one FLOW installation with multiple MagNA Pure Instruments (up to 3) and qPCR Instruments (up to 5). This option is supported in linear and nonlinear workflows and only one FLOW Software license is required. You may also combine FLOW Ultra with FLOW Fusion.

2 3

Redesign your lab with the FLOW Solution

The FLOW Solution is a highly flexible modular, semi-automated data and sample workflow. It seamlessly processes various sample and tube types in a completely traceable barcoded manner from primary sample handling, NA extraction and PCR setup to qPCR. LIMS connectivity, middleware software management and a clever IT structure enables a smooth data transition without leaving room for manual errors. The modular instrument and assay concept allows you to truly develop your lab, your way.

FLOW Classic

FLOW FlexFLOW Flex is the minimal instrument configuration necessary to be supported by the FLOW Software. It consists of a PCR Setup Instrument, a MagNA Pure 96 and a Roche qPCR Instrument. FLOW Flex operates with one pipetting robot used for both Primary Sample Handling and PCR Setup. It generates a non linear sample workflow with dual use of the Roche Liquid Handling Platform (PCR Setup Instrument).

FLOW Classic is the same instrumentation configuration as FLOW Flex with an additional pipetting robot. This could be either a Primary Sample Handling Instrument or a PCR Setup Instrument. In this configuration, the workflow is linear and has dedicated tasks for each pipetting robot.

Expand your possibilities

There are two ways to grow your FLOW Solution: Install an additional FLOW Flex or FLOW Classic or add extraction and amplification systems to an existing workflow. Here are some examples for options:

FLOW Ultra

FLOW FusionFLOW Fusion is a software application that allows you to run two separate FLOW installations in a coordinated manner. It allows for independent loading of samples and generates more flexible workflow options. This software tool operates both, linear and nonlinear workflows. To support this configuration a FLOW Software Additional License is necessary. FLOW Fusion and FLOW Software Additional License will be available at the launch of FLOW Software version 2.1.

FLOW Ultra is a configuration which expands one FLOW installation with multiple MagNA Pure Instruments (up to 3) and qPCR Instruments (up to 5). This option is supported in linear and nonlinear workflows and only one FLOW Software license is required. You may also combine FLOW Ultra with FLOW Fusion.

2 3

Primary Sample Handling Instrument

Purpose Primary Sample Handling for the Roche MagNA Pure 96 SystemTransfer of samples from common tube types into the Roche MagNA Pure 96 Processing Cartridges

Type Benchtop instrument

Dimensions 112.4 × 90.3 × 100.6 cm (W × D × H)

Weight 163 kg

Run time Transfer of 96 samples from sample tubes to plates in approximately 20 minutes depending on sample material

Setup time Up to 15 minutes to manually place samples, tips, and plates onto loading trayStage loading, including barcode reading is performed automatically

Sample types Without pretreatment: Whole blood, serum, plasma, cell suspension (resuspended in PBS), saliva, urineWith pretreatment: BAL, stool, swab eluates, CSF, sputum

Supported primary/secondary tube types

15 × 100 mm, 15 × 75 mm, 12 × 100 mm, 12 × 75 mmAdapters to fit FLOW Tubes (2 ml)Can support up to 256 samples per run

Plate capacity 1 Processing FLOW Cartridge carrier for maximum 3 MagNA Pure 96 Processing Cartridges

Sample volume 50 – 500 µl, 1,000 µl for 48 samples only

Liquid handling One pipetting arm with 4 independently controlled air displacement channels

Liquid level detection Capacitive liquid level detection (cLLD)

Min. pipetting volume 50 µl for sample, 20 µl for internal control

Barcode scanning Barcode scanner mounted on the autoload slide for tips and tubesBarcode scanner fixed on the instrument deck for plates

System sterility UV light

Disposables

Tips Tip High Vol, 1 ml with Filter

Plates 96 well MagNA Pure 96 Processing Cartridges, Note: Maximum volume depends on used sample volume

Tubes FLOW Tubes (2ml)

FLOW Primary Sample Handling Instrument

• Works for different sample types • Flexibly accepts primary & secondary sample tubes • Pipets sample into MagNA Pure 96 Processing Cartridge • Offers clot detection and a closed housing • Comprehensive process surveillance • GMP manufactured by Hamilton

MagNA Pure 96 Instrument

Purpose The MagNA Pure 96 System purifies DNA, RNA, and viral nucleic acids from a wide range of starting materials using magnetic glass particle technology (MGP)


Dimensions 136 × 81.5 × 100 cm (W × D × H)

Weight 235 kg

Run time Approximately 50 to 60 minutes for 200 μl sample volumesApproximately 80 to 90 minutes for 500 μl sample volumes

Racks Drawer-like loading racks for extraction reagents

Sample number 1 to 96 reactions per run

Plate capacity 1 for MagNA Pure 96 Processing Cartridges1 for MagNA Pure 96 Output Plates

Sample volume 50 to 500 μl with 96 reactions per run, 1,000 µl for 48 samples only

Elution volume 50 to 200 µl

Liquid handling Two robotic arms:1. Reagent head with four individually controlled fluid channels2. Process head with a 96-nozzle pipette head (CO-RE tip technology)

Liquid pressure detection Liquid pressure sensor

Barcode scanning Internal and external barcode scanner

System sterility UV lights

Disposables

Tips MagNA Pure 96 Filter Tips (1,000 µl)

Plates MagNA Pure 96 Processing CartridgesMagNA Pure 96 Output Plates

Reagents

System fluid MagNA Pure System Fluid Internal Container, for 2 L, orMagNA Pure System Fluid External Container, for 5 L

Prefilled, ready-to-use reagents

MagNA Pure 96 DNA and Viral NA Small Volume Kit (CE-IVD)MagNA Pure 96 DNA and Viral NA Large Volume Kit (CE-IVD)MagNA Pure 96 Cellular RNA Large Volume Kit (For life science research only. Not for use in diagnostic procedures.)


• Offers high speed nucleic acid isolation for up to 96 samples in less than 1 hour

• Flexibly accepts more than 10 different sampletypes within one run

• Provides for result safety through run surveillance and tracking

• Consistent sample handling with excellent intrarun, inter-run, and lot-to-lot precision

• GMP manufactured, CE-IVD registered

4 5

Primary Sample Handling Instrument

Purpose Primary Sample Handling for the Roche MagNA Pure 96 SystemTransfer of samples from common tube types into the Roche MagNA Pure 96 Processing Cartridges



Weight 163 kg

Run time Transfer of 96 samples from sample tubes to plates in approximately 20 minutes depending on sample material

Setup time Up to 15 minutes to manually place samples, tips, and plates onto loading trayStage loading, including barcode reading is performed automatically

Sample types Without pretreatment: Whole blood, serum, plasma, cell suspension (resuspended in PBS), saliva, urineWith pretreatment: BAL, stool, swab eluates, CSF, sputum

Supported primary/secondary tube types

15 × 100 mm, 15 × 75 mm, 12 × 100 mm, 12 × 75 mmAdapters to fit FLOW Tubes (2 ml)Can support up to 256 samples per run

Plate capacity 1 Processing FLOW Cartridge carrier for maximum 3 MagNA Pure 96 Processing Cartridges

Sample volume 50 – 500 µl, 1,000 µl for 48 samples only

Liquid handling One pipetting arm with 4 independently controlled air displacement channels


Min. pipetting volume 50 µl for sample, 20 µl for internal control

Barcode scanning Barcode scanner mounted on the autoload slide for tips and tubesBarcode scanner fixed on the instrument deck for plates

System sterility UV light

Disposables

Tips Tip High Vol, 1 ml with Filter

Plates 96 well MagNA Pure 96 Processing Cartridges, Note: Maximum volume depends on used sample volume


FLOW Primary Sample Handling Instrument

• Works for different sample types • Flexibly accepts primary & secondary sample tubes • Pipets sample into MagNA Pure 96 Processing Cartridge • Offers clot detection and a closed housing • Comprehensive process surveillance • GMP manufactured by Hamilton


Purpose The MagNA Pure 96 System purifies DNA, RNA, and viral nucleic acids from a wide range of starting materials using magnetic glass particle technology (MGP)


Dimensions 136 × 81.5 × 100 cm (W × D × H)

Weight 235 kg

Run time Approximately 50 to 60 minutes for 200 μl sample volumesApproximately 80 to 90 minutes for 500 μl sample volumes

Racks Drawer-like loading racks for extraction reagents

Sample number 1 to 96 reactions per run

Plate capacity 1 for MagNA Pure 96 Processing Cartridges1 for MagNA Pure 96 Output Plates

Sample volume 50 to 500 μl with 96 reactions per run, 1,000 µl for 48 samples only

Elution volume 50 to 200 µl

Liquid handling Two robotic arms:1. Reagent head with four individually controlled fluid channels2. Process head with a 96-nozzle pipette head (CO-RE tip technology)

Liquid pressure detection Liquid pressure sensor

Barcode scanning Internal and external barcode scanner

System sterility UV lights

Disposables

Tips MagNA Pure 96 Filter Tips (1,000 µl)

Plates MagNA Pure 96 Processing CartridgesMagNA Pure 96 Output Plates

Reagents

System fluid MagNA Pure System Fluid Internal Container, for 2 L, orMagNA Pure System Fluid External Container, for 5 L

Prefilled, ready-to-use reagents

MagNA Pure 96 DNA and Viral NA Small Volume Kit (CE-IVD)MagNA Pure 96 DNA and Viral NA Large Volume Kit (CE-IVD)MagNA Pure 96 Cellular RNA Large Volume Kit (For life science research only. Not for use in diagnostic procedures.)


• Offers high speed nucleic acid isolation for up to 96 samples in less than 1 hour

• Flexibly accepts more than 10 different sampletypes within one run

• Provides for result safety through run surveillance and tracking

• Consistent sample handling with excellent intrarun, inter-run, and lot-to-lot precision

• GMP manufactured, CE-IVD registered

4 5

Roche Liquid Handling Instrument*

Purpose Primary Sample Handling and/or PCR Setup



Weight 180 kg

Setup time Up to 15 minutes

Run time Dependent on number of samples, complexity of reaction mix setup, and number of total reactions; 30-50 minutes for most common applications

Plate capacity 5 positions for LightCycler® 480 Multiwell PCR Plates 3 positions for the MagNA Pure 96 Output Plates 2 positions for RT reaction plates (requires external incubation)

Automated master mix preparation

For ready-to-use master mixes, detection mixes, or single components Up to 3 components (enzyme, water, additional component) can be combined for master mix reactions Up to 3 components (forward primer, reverse primer, probe) can be combined for reaction mix preparationSupports up to 21 components to create one PCR master mix

Liquid handling 8 independent 1 ml pipetting channels


Minimal pipetting volume 5 μl to empty well, 2 μl to partially filled well in 384-well, and 5 μl to empty well, 5 μl to partially filled well in 96-well

Barcode scanning Barcode scanner mounted on the autoload slide for tips and tubes Barcode scanner fixed on the instrument deck for plates

Disposables

Tips 1 ml, 300 µl and 50 µl filter tips

Plates MagNA Pure 96 Output PlatesLightCycler® 480 Multiwell Plates 96 wells and 384 wells (Specific plates with 4 barcodes only)


FLOW PCR Setup Instrument

• Data driven pipetting • Highly flexible PCR Setup

• 96 and 384 well plate capability • Automated master mix creation

• GMP manufactured by Hamilton • Supports FLOW Primary Sample Handling

* All specs described here are relevant for PCR Setup only. For Primary Sample Handling specifications refer to Primary Sample Handling Instrument specification on page 4.

Roche qPCR Instrument

Purpose qPCR reaction in 96 or 384 PCR plate formats



Weight 55 kg

Run time < 40 min. for 40 cycles in 384-well plate format40 cycles, 2-step protocols: < 40 min. (384)40 cycles, 2-step protocols: < 60 min. (96)

Reactions per run Up to 96 or 384 reactions per run (384 well block available for LightCycler® 480 only)

Probe /assay format Intercalating dyes and Hydrolysis probes

Multiplexing capability 6x

Cq uniformity SD < 0.2

Temperature accuracy ± 0.2°C of target temperature

FLOW Reaction volumes 7 –20 μl (384)/10 –100 μl (96)

Analysis type Absolute quantification based on 2nd derivative maximum method

Disposables

Plates LightCycler® 480 Multiwell Plates 96/384 wells (Specific plates with 4 barcodes only)


• Speeds through 40 cycles in 40 – 60 minutes • Well established industry standard with

thousands of placements worldwide • Top notch in:

• Temperature accuracy • Sensitivity

• Connection of up to five 96-well or 384-well Roche qPCR Instruments

• GMP manufactured

6 7

Roche Liquid Handling Instrument*

Purpose Primary Sample Handling and/or PCR Setup



Weight 180 kg

Setup time Up to 15 minutes

Run time Dependent on number of samples, complexity of reaction mix setup, and number of total reactions; 30-50 minutes for most common applications

Plate capacity 5 positions for LightCycler® 480 Multiwell PCR Plates 3 positions for the MagNA Pure 96 Output Plates 2 positions for RT reaction plates (requires external incubation)

Automated master mix preparation

For ready-to-use master mixes, detection mixes, or single components Up to 3 components (enzyme, water, additional component) can be combined for master mix reactions Up to 3 components (forward primer, reverse primer, probe) can be combined for reaction mix preparationSupports up to 21 components to create one PCR master mix

Liquid handling 8 independent 1 ml pipetting channels


Minimal pipetting volume 5 μl to empty well, 2 μl to partially filled well in 384-well, and 5 μl to empty well, 5 μl to partially filled well in 96-well

Barcode scanning Barcode scanner mounted on the autoload slide for tips and tubes Barcode scanner fixed on the instrument deck for plates

Disposables

Tips 1 ml, 300 µl and 50 µl filter tips

Plates MagNA Pure 96 Output PlatesLightCycler® 480 Multiwell Plates 96 wells and 384 wells (Specific plates with 4 barcodes only)


FLOW PCR Setup Instrument

• Data driven pipetting • Highly flexible PCR Setup

• 96 and 384 well plate capability • Automated master mix creation

• GMP manufactured by Hamilton • Supports FLOW Primary Sample Handling

* All specs described here are relevant for PCR Setup only. For Primary Sample Handling specifications refer to Primary Sample Handling Instrument specification on page 4.


Purpose qPCR reaction in 96 or 384 PCR plate formats



Weight 55 kg

Run time < 40 min. for 40 cycles in 384-well plate format40 cycles, 2-step protocols: < 40 min. (384)40 cycles, 2-step protocols: < 60 min. (96)

Reactions per run Up to 96 or 384 reactions per run (384 well block available for LightCycler® 480 only)

Probe /assay format Intercalating dyes and Hydrolysis probes

Multiplexing capability 6x

Cq uniformity SD < 0.2

Temperature accuracy ± 0.2°C of target temperature

FLOW Reaction volumes 7 –20 μl (384)/10 –100 μl (96)

Analysis type Absolute quantification based on 2nd derivative maximum method

Disposables

Plates LightCycler® 480 Multiwell Plates 96/384 wells (Specific plates with 4 barcodes only)


• Speeds through 40 cycles in 40 – 60 minutes • Well established industry standard with

thousands of placements worldwide • Top notch in:

• Temperature accuracy • Sensitivity

• Connection of up to five 96-well or 384-well Roche qPCR Instruments

• GMP manufactured

6 7

Run schedule for FLOW Classic

FLOW SoftwareApplication Qualitative and Quantitative Analysis

Run time used PCR Approximately 4 hours depending on number and type of sample materials used, complexity of PCR setup, and used PCR profile

Throughput Up to 256 sample extractions per run (with 3 MagNA Pure 96 Instruments)

Assay flexibility Detect a multitude of targets per sample and per plate; use one or multiple plates for separate run profiles

Configurations FLOW Classic, FLOW Flex, FLOW Fusion and FLOW Ultra (Check page 2 and 3 for details)

Hands-on time 20–30 sec. per sample for the complete FLOW Solution

Daily maintenance and cleaning activities approximately 15min. (for all instruments)

FLOW Software Manages the entire workflow Manages information exchange between all modules Communicates with LIS system: • LIMS download of sample information • LIMS download of test requests • Generates work lists • Reports test results back to LIMS

FLOW setup Separate setup for all benchtop instruments and FLOW control unit possible (Multi room concept)

Overlapping runs When purification of first order is finished, a new order can be started approximately every 1.5 hours. This results in approximately 4 overlapping FLOW runs per day for FLOW Classic and 3 for FLOW Flex.

Retests Yes, from samples and eluates

Reflex tests Yes, from already tested eluates

The FLOW Solution is used for many applications. Here is an example of a multiplex PCR for 3 targets and an internal control.

Experimental Goal: Detect 3 different targets and internal control with quadruplex PCR.

Experimental Set Up: A multiplex assay with 3 different target genes and one internal control was designed. Negative process and positive controls were used to validate the result.

Fig.2: FLOW Software display for Target 1,2,3 and internal control. Graphs shows target gene samples (red) controls (green).

Applications and performance data

Materials Used: Primary sample material was suspended in S.T.A.R buffer. Extraction was done with the MagNA Pure 96 DNA Viral NA Small Volume Kit, using the Pathogen Universal 200 protocol. qPCR was performed with the RNA Process Control Kit which includes the LightCycler® Multiplex RNA Master.

Results and Data: Positive samples (Red curves, yellow circles) were found for all targets. Controls are in green. For each target positive samples were detected, showing the FLOW Solution can be run in a multiplex set up without crosstalk (Fig.2).

9

FLOW Software: Connect your lab, your way

1. Qualitative Detection of multiple targets

*A run depends on number and type of sample materials used, complexity of PCR setup, and PCR profile.

Complete one FLOW process in approximately 4 hours. When the purification of the first run is finished, a new workflow can be started (approximately every 1.5 hours), resulting in 4 overlapping FLOW runs* per day (see Fig.1).

4 runs on the FLOW Classic Solution every 8 hours

WORKING DAY8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00

1

2

3

PSHSample

Preparation PCR Setup PCR 4

PSHSample

Preparation PCR Setup PCR

PSHSample


PSHSample


Fig.1: Run schedule for FLOW Classic.

Run schedule for FLOW Classic

FLOW SoftwareApplication Qualitative and Quantitative Analysis

Run time used PCR Approximately 4 hours depending on number and type of sample materials used, complexity of PCR setup, and used PCR profile

Throughput Up to 256 sample extractions per run (with 3 MagNA Pure 96 Instruments)

Assay flexibility Detect a multitude of targets per sample and per plate; use one or multiple plates for separate run profiles

Configurations FLOW Classic, FLOW Flex, FLOW Fusion and FLOW Ultra (Check page 2 and 3 for details)

Hands-on time 20–30 sec. per sample for the complete FLOW Solution

Daily maintenance and cleaning activities approximately 15min. (for all instruments)

FLOW Software Manages the entire workflow Manages information exchange between all modules Communicates with LIS system: • LIMS download of sample information • LIMS download of test requests • Generates work lists • Reports test results back to LIMS

FLOW setup Separate setup for all benchtop instruments and FLOW control unit possible (Multi room concept)

Overlapping runs When purification of first order is finished, a new order can be started approximately every 1.5 hours. This results in approximately 4 overlapping FLOW runs per day for FLOW Classic and 3 for FLOW Flex.

Retests Yes, from samples and eluates

Reflex tests Yes, from already tested eluates

The FLOW Solution is used for many applications. Here is an example of a multiplex PCR for 3 targets and an internal control.

Experimental Goal: Detect 3 different targets and internal control with quadruplex PCR.

Experimental Set Up: A multiplex assay with 3 different target genes and one internal control was designed. Negative process and positive controls were used to validate the result.

Fig.2: FLOW Software display for Target 1,2,3 and internal control. Graphs shows target gene samples (red) controls (green).

Applications and performance data

Materials Used: Primary sample material was suspended in S.T.A.R buffer. Extraction was done with the MagNA Pure 96 DNA Viral NA Small Volume Kit, using the Pathogen Universal 200 protocol. qPCR was performed with the RNA Process Control Kit which includes the LightCycler® Multiplex RNA Master.

Results and Data: Positive samples (Red curves, yellow circles) were found for all targets. Controls are in green. For each target positive samples were detected, showing the FLOW Solution can be run in a multiplex set up without crosstalk (Fig.2).

9

FLOW Software: Connect your lab, your way

1. Qualitative Detection of multiple targets

*A run depends on number and type of sample materials used, complexity of PCR setup, and PCR profile.

Complete one FLOW process in approximately 4 hours. When the purification of the first run is finished, a new workflow can be started (approximately every 1.5 hours), resulting in 4 overlapping FLOW runs* per day (see Fig.1).

4 runs on the FLOW Classic Solution every 8 hours

WORKING DAY8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00

1

2

3

PSHSample

Preparation PCR Setup PCR 4

PSHSample


PSHSample


PSHSample


Fig.1: Run schedule for FLOW Classic.

Graph 1: Standard curve as displayed in the Roche qPCR instrument with 7, 10-fold dilutions.

Fig.3: Standard curve generated by the FLOW Software from the data shown in Graph 1.

Target Standards Std Dev Mean Min Max DeltapCP-1 2,00E+04 0,37 33,69 33,36 34,09 0,73

2,00E+05 0,10 30,22 30,14 30,32 0,18

2,00E+06 0,05 26,63 26,58 26,66 0,08

2,00E+07 0,02 23,16 23,14 23,18 0,04

2,00E+08 0,13 19,86 19,77 20,01 0,24

2,00E+09 0,04 16,33 16,30 16,37 0,07

2,00E+10 0,06 13,32 13,25 13,38 0,13

Tab.1: Cq values for the standard curve displayed in Fig.2 and Graph 1 generated with FLOW Classic. Standards are shown in copies/ml. Each standard was measured in separate isolated triplicates.

Target pCP-1 Sample PSU ReplicatesExtractionReplicates

Sample 1 28,77 28,77

Sample 2 28,93 29,01

Sample 3 28,57 28,44

Sample 4 28,80 28,86

Sample 5 28,70 28,78

Tab.2: Samples quantified with the standard curve in Fig.2. Rows are pipetting replicates on the PSU, while columns are replicate samples on a complete FLOW run.

3. Inter- and intra-run reproducibility

Experiment Goal: Check for intra- and inter-run reproducibility data for positive and negative results.

Experimental Set Up: The following experiments were performed with FLOW Flex. Four consecutive runs on the same instruments were setup to check the reproducibility performance of the FLOW Solution. The first run was done with 95 negative samples and one positive control. Two follow up runs with a checkerboard setup were accomplished with 48 negative and 48 positive samples. Finally, the first run was repeated.

Materials Used: Negative samples: K3-EDTA negative pooled human plasma. Positive samples: K3-EDTA negative pooled human plasma spiked with pCP-plasmid. Extraction was done with the MagNA Pure 96 DNA Viral NA Small Volume Kit, using the 200µl universal pathogen protocol. qPCR was performed with the LightCycler® 480 Probes Master.

PCR Plate ID N Std Dev Mean Min Max5120646 48 0,18 17,88 17,44 18,19

5120647 48 0,20 17,92 17,57 18,29

All 96 0,19 17,9 17,44 18,29

Tab.3: Positve results of the 2 checkerboard runs from Fig.5 and Fig.6.

Fig.4: First run with one positive control in A1 and all the rest negative. Fig.5: Second run with controls in A1 (pos) and B1 (neg).

Fig.6: Third run with controls in A1 (pos) and B1 (neg). Fig.7: Forth run with one positive control in A1 and all the rest negative.

Results: Negative samples stayed negative (Fig.4 and Fig.7). No cross-contamination was found on either intra- or inter- run data.

Positive samples showed a very high reproducibility with a standard deviation of 0,20 and 0,18 respectively (Fig.5 and Fig.6). The run to run difference was 0,04 Cq on the mean (Tab.3).

10 11

Quantification of targets is usually done with a standard curve generated on the PCR level only. In the following experiment a standard curve was generated by extracting each standard individually in triplicates. With this approach the standard deviation of the whole workflow is measured rather than on the PCR level only.

Experimental Goal: Show quantification efficiency of the FLOW Solution.

Materials used: Negative samples: K3-EDTA negative pooled human plasma. Positive samples: K3-EDTA negative pooled human plasma spiked with pCP- plasmid in the concentrations given in table 1. Extraction was done with the MagNA Pure 96 DNA Viral NA Small Volume Kit, using the Pathogen Universal 200 protocol. qPCR was performed with the LightCycler® 480 Probes Master.

Experiment Setup: All standards were used in triplicates with separate extraction on each; a pool was split to 5 samples (Tab.2). Samples together with standards were run on FLOW Classic.

Results & Conclusion: A standard curve profile with a low error (0,0147) and a high efficiency (1,988) (Fig.3, Graph 1) was generated. Standard deviations (mean 0,11) were very low, which documents an excellent precision of the whole process (Tab.1).

Separate extracted samples could be quantified at a late Cq with high reproducibility. In summary FLOW offers a reliable way to quantify samples.

Consistent and reliable results are key for each workflow application. In this series of experiments, the reproducibility of data generated by the FLOW Solution was evaluated.

2. Quantitative Detection of a single target

Graph 1: Standard curve as displayed in the Roche qPCR instrument with 7, 10-fold dilutions.

Fig.3: Standard curve generated by the FLOW Software from the data shown in Graph 1.

Target Standards Std Dev Mean Min Max DeltapCP-1 2,00E+04 0,37 33,69 33,36 34,09 0,73

2,00E+05 0,10 30,22 30,14 30,32 0,18

2,00E+06 0,05 26,63 26,58 26,66 0,08

2,00E+07 0,02 23,16 23,14 23,18 0,04

2,00E+08 0,13 19,86 19,77 20,01 0,24

2,00E+09 0,04 16,33 16,30 16,37 0,07

2,00E+10 0,06 13,32 13,25 13,38 0,13

Tab.1: Cq values for the standard curve displayed in Fig.2 and Graph 1 generated with FLOW Classic. Standards are shown in copies/ml. Each standard was measured in separate isolated triplicates.

Target pCP-1 Sample PSU ReplicatesExtractionReplicates

Sample 1 28,77 28,77

Sample 2 28,93 29,01

Sample 3 28,57 28,44

Sample 4 28,80 28,86

Sample 5 28,70 28,78

Tab.2: Samples quantified with the standard curve in Fig.2. Rows are pipetting replicates on the PSU, while columns are replicate samples on a complete FLOW run.

3. Inter- and intra-run reproducibility

Experiment Goal: Check for intra- and inter-run reproducibility data for positive and negative results.

Experimental Set Up: The following experiments were performed with FLOW Flex. Four consecutive runs on the same instruments were setup to check the reproducibility performance of the FLOW Solution. The first run was done with 95 negative samples and one positive control. Two follow up runs with a checkerboard setup were accomplished with 48 negative and 48 positive samples. Finally, the first run was repeated.

Materials Used: Negative samples: K3-EDTA negative pooled human plasma. Positive samples: K3-EDTA negative pooled human plasma spiked with pCP-plasmid. Extraction was done with the MagNA Pure 96 DNA Viral NA Small Volume Kit, using the 200µl universal pathogen protocol. qPCR was performed with the LightCycler® 480 Probes Master.

PCR Plate ID N Std Dev Mean Min Max5120646 48 0,18 17,88 17,44 18,19

5120647 48 0,20 17,92 17,57 18,29

All 96 0,19 17,9 17,44 18,29

Tab.3: Positve results of the 2 checkerboard runs from Fig.5 and Fig.6.

Fig.4: First run with one positive control in A1 and all the rest negative. Fig.5: Second run with controls in A1 (pos) and B1 (neg).

Fig.6: Third run with controls in A1 (pos) and B1 (neg). Fig.7: Forth run with one positive control in A1 and all the rest negative.

Results: Negative samples stayed negative (Fig.4 and Fig.7). No cross-contamination was found on either intra- or inter- run data.

Positive samples showed a very high reproducibility with a standard deviation of 0,20 and 0,18 respectively (Fig.5 and Fig.6). The run to run difference was 0,04 Cq on the mean (Tab.3).

10 11

Quantification of targets is usually done with a standard curve generated on the PCR level only. In the following experiment a standard curve was generated by extracting each standard individually in triplicates. With this approach the standard deviation of the whole workflow is measured rather than on the PCR level only.

Experimental Goal: Show quantification efficiency of the FLOW Solution.

Materials used: Negative samples: K3-EDTA negative pooled human plasma. Positive samples: K3-EDTA negative pooled human plasma spiked with pCP- plasmid in the concentrations given in table 1. Extraction was done with the MagNA Pure 96 DNA Viral NA Small Volume Kit, using the Pathogen Universal 200 protocol. qPCR was performed with the LightCycler® 480 Probes Master.

Experiment Setup: All standards were used in triplicates with separate extraction on each; a pool was split to 5 samples (Tab.2). Samples together with standards were run on FLOW Classic.

Results & Conclusion: A standard curve profile with a low error (0,0147) and a high efficiency (1,988) (Fig.3, Graph 1) was generated. Standard deviations (mean 0,11) were very low, which documents an excellent precision of the whole process (Tab.1).

Separate extracted samples could be quantified at a late Cq with high reproducibility. In summary FLOW offers a reliable way to quantify samples.

Consistent and reliable results are key for each workflow application. In this series of experiments, the reproducibility of data generated by the FLOW Solution was evaluated.

2. Quantitative Detection of a single target

DEVELOP YOUR LAB, YOUR WAYThe FLOW Solution: Expand your potential today

Technical brochure

www.flow.roche.com

Published by Roche Molecular Systems, Inc. 4300 Hacienda Drive Pleasanton, CA 94588 USA

© 2015 Roche Molecular Systems Inc.

The FLOW Solution is not available in all territories due to different national regulations.This document is not intended for use in the USA. For general laboratory use.

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For general laboratory use. DEVELOP YOUR LAB, YOUR WAY€¦ · · 2015-10-06instrument and assay concept allows you to truly develop your lab, your way. FLOW Classic ... Liquid