ViiA™ 7 Real-Time PCR System with 384-Well Block - FAQs

In the ViiA 7 Real-Time PCR System, please follow the instructions found in the Software Licensing Quick Reference Guide at https://fnoclient.gss.tf/quick-start-guide to activate or renew a license. If you have a license key associated with a computer that is no longer in use, please follow the instructions to "Transfer a license to a different user or computer" in the above guide.

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This error can occur when the run was started from a corrupt template file or if the file name is very long or has illegal characters. Delete any templates that were created in v1.1 or earlier. Upgrade your software to the most current version. Recreate your templates in v1.2 or later. Only use templates created in v1.2 or later for any future runs. If the file name is very long, try using the “Save As” feature to resave the file with a shorter name. If this does not fix the problem, please send your file to techsupport@thermofisher.com for further assistance.

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After exporting data into .xls format, the Quantity or Ct values may not be present. From your file, Select ‘Export‘ and click on ‘Load Export Set' and then ‘OK' and ‘OK'. Then click on ‘Start Export'. The Quantity or Ct values should now be present in the exported file.

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This error is caused by a corrupt template or when the ‘Open Run Method' button was selected to start the run. To avoid this, do not use corrupt templates or run files from previous software versions. If you have v.1.1 or earlier, please upgrade to the most current version. If you are using templates created from v.1.1 or earlier, please delete and recreate them in v1.2 or above. New run files created from v1.2 can use the ‘Open Run Method' button.
If you have a file that gives you this error, please send your data file (*.eds) to techsupport@thermofisher.com for assistance.

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The calibration files are stored on the ViiA 7 instrument itself; therefore, when you need to perform a new calibration, you will have to delete the old calibration files. From the instrument console, go to the ViiA 7 instrument touchscreen and select ‘Collect Results'. You should then see a list of files stored on the instrument. Manually select a calibration file such as “Fast-96-cal”, and press the ‘Delete' button to remove the old calibration file. Occasionally, users cannot delete the file off the instrument and may encounter this error: “Some experiments did not collect results therefore cannot be deleted.”. In these situations, launch the software, go to ‘maintenance', manage files, and download the calibration file. The status of the experiment should be displayed as ‘complete'. Next, delete the old calibration file and rerun the calibration.

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After launching the ViiA 7 system software, go to the Maintenance Manager. Check if the ViiA 7 system icon is present in the “On the Network” window. If not, click on the “Refresh” button to the left of “Remove from My Instruments”.

If you still do not see the ViiA 7 icon for the following reasons:
- There is an icon but you cannot add it into the “My Instruments” area
- There is a red “X” on the instrument
- A message says “Disconnected” at the bottom of the window with a red “X” in it then you will need to add the instrument to the “My Instruments” area. Click on the wrench icon in the upper right-hand corner of the icon as pictured below. You may get a message that says that the firmware is older than the one you are trying to run. If appropriate, choose to download the new firmware and let it progress to finish. Do not turn off the instrument during this update. After the firmware update is completed, restart the instrument to check the ViiA 7 system icon in ‘On the Network' area.

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For genotyping data, we recommend TaqMan Genotyper Software. For relative quantitation, we recommend ExpressionSuite Software.

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Yes. The ViiA 7 system can collect data at multiple steps in the amplification stage. You will need to turn on the data collection for every step of interest. In the analysis, only one set of data can be displayed at a time. To change the data set used for analysis, from your data file go to Analysis Settings then Ct Settings and change the dropdown under ‘Data Step Selection'.

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The following volumes are supported for each instrument block:

- 96-well standard (10-100 µL reactions)
- 96-well Fast (10-30 µL reactions)
- 384-well (5-20 µL reactions)
- TaqMan Array Micro Fluidic Cards (approximately 1 µL reactions)

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The ViiA 7 Real-Time PCR System can store up to 100 runs on the instrument itself.

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You will need an oligo sequence with the custom dye but without the quencher molecule. For each custom dye, prepare samples in a concentration range of 100-2,000 nM, choosing a 2-3-fold difference in dilution points. Use either the background buffer provided in the calibration kit or your own buffer to dilute the oligo. A full plate is not needed; see the example below.

1. Set up a dummy run using the ‘Standard Curve' option. Alter the thermal profile so that it simply ramps to 60 degrees C with a 2 min hold. Ensure that the filters of interest are selected.
2. When the run is complete, export and examine the raw data. Select the concentration to use by finding the dilution that will give you an acceptable signal in the following ranges:
- For a 384-well plate: between 400,000 and 1,200,000
3.For a 96-well plate: between 1,400,000 and 4,300,000
4.Create a full plate of the dye using the selected concentration and run the custom dye calibration as normal, using 20 µL. (Refer to the ViiA 7 Real-Time PCR System Maintenance Guide, “Creating a custom dye plate”, for full details.)

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In a touchdown PCR experiment, you will change either the temperature or the time of a particular PCR step with every cycle. Most commonly, the annealing temperature is adjusted throughout the experiment, such that the specificity is higher in the early cycles and the efficiency in the later cycles. In this example, we will set the method to do the following:

1. Go to File then New Experiment then Advanced Setup. Fill out the relevant options as you normally would.
2. Go to the Run Method under the ‘Setup' section and you should see the Graphical View of your thermal profile. Check the box next to ‘Enable AutoDelta'. You should see some grey triangles appear next to the Temperature and Time at every step in the Cycling Stage. (Note: If you want to start the changes at a later cycle, set this here under ‘Starting Cycles'.)
3.A new window called ‘AutoDelta Settings' will open up. Select the appropriate options and click ‘Save Setting'. You will then see a green triangle show up next to the parameter you changed. Your new method has now been applied.

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You can create your own background calibration plate using the directions in the ViiA 7 Real-Time PCR System Maintenance Guide (Appendix C) http://tools.thermofisher.com/content/sfs/manuals/4442661C.pdf. For all other calibration plates, you can purchase a calibration kit, or the individual plates, as required (http://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-reagents/real-time-calibration-kits/viia-7-system-calibration-kits.html).

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The calibration plates can be stored and reused three times for up to 1 year after you first open them, so make sure to return them to their original packaging and return them to -20 degrees C storage until the next use. (Note: Due to the small volumes, calibration array cards cannot be reused. Please discard them after use.) If needed, you can make your own background plate using deionized water. Please follow the directions in the ViiA 7 Real-Time PCR System Installation, Networking, and Maintenance Guide for more details at http://tools.thermofisher.com/content/sfs/manuals/4442661C.pdf.

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The uniformity calibration generates data that allow the software to compensate for the physical effects of the ViiA 7 system filters using the ROI plate. The normalization calibration generates factors that the software uses when comparing data from multiple ViiA 7 instruments within a study. Two normalization plates are provided in the calibration kit, one containing FAM/ROX dyes and the other with VIC/ROX dyes.

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The recommended maintenance schedule and calibration instructions can be found in the User Guide here: http://tools.thermofisher.com/content/sfs/manuals/4442661C.pdf
In brief, the background calibration is recommended to be done every month. All other calibrations (pure dye, ROI, uniformity, and normalization) are recommended to be done every six months.

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User Bulletin v1.2.1 has specific directions for how to renew your software license. Please search the term '4474347' on our website. The renewal directions apply to the ViiA 7, QuantStudio 6, QuantStudio 7, and QuantStudio 12K Flex systems.

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You can check your license status by opening the instrument software and going to Tools --> License Central. You will see the license status and the expiration date.

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Yes, on the QuantStudio 12K Flex system you can see the real time amplification plots on the touchscreen. On other systems, you will only be able to see the amplification plots in real-time on the connected computer.

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If using a mastermix in which ROX dye is provided in a separate tube, please add it to 50 nM (in the final reaction well).

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If you need to move your ViiA 7 system software to another computer, log in to the Thermo Fisher Scientific licensing site at https://thermofisher1.flexnetoperations.com/flexnet/operationsportal/logon.do. Instructions for transferring licenses to a new computer are located in the Quick Reference Guide (https://fnoclient.gss.tf/quick-start-guide) under the "Transfer a license to a different user or computer" section.

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The normalization calibration generates factors that the software uses when comparing data from multiple instruments.

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The ViiA 7 can store up to 100 data files. When the instrument data storage becomes full, simply touch the 'Collect Results' button and select the files you want to delete to free up more space.

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It is possible to import a standard curve if you have either the ViiA 7 or QuantStudio 12K Flex systems. In the ViiA 7 or QuantStudio instrument software, open the data file and go to 'Analysis Settings'. Click on the 'Standard Curve Settings' tab. You will see an option to 'Use a standard curve imported from another experiment'. Choose this, then click on "Import". Browse to the proper data file and click "Select".

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The ViiA 7 is calibrated for the following dyes: FAM, VIC, ROX, SYBR, NED, and TAMRA. In addition, it can be used with custom dyes that excite between 455 and 672 nm and read between 505 and 723 nm.

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The StepOne, StepOnePlus, ViiA 7, and QuantStudio 12K Flex systems can all be run directly from the instrument touchscreen and don't need a separate computer to operate them.

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All run files have an .SDS or .EDS extension. A Relative Quantification study will have an .SDM or .EDM extension and a template will have an .SDT or .EDT extension. The software for StepOnePlus, ViiA 7, and 7500 2.0.x software have .EDS, .EDT, or .EDM extensions; all other software have .SDS, .SDT, or .SDM extensions.

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The systems most often vary by the light source and filter sets. The 7000, 7300, 7500, and 7500 Fast systems utilize a halogen lamp as the excitation source and emission filters to detect the emission fluorescence. The 7000 and 7300 systems both have one excitation and four emission filters, while the 7500 and 7500 Fast systems have five excitation and five emission filters. Five excitation and five emission filters allow the 7500 and 7500 Fast systems to detect dyes in the far red range. The 7700 and 7900HT systems utilize an argon ion laser as the excitation source and the instrument scans across all wavelengths between 520 nm to 610 nm.

The 7500, 7500 Fast and 7900HT systems also offers the ability to upgrade to the Fast chemistry; typical real-time runs take about 30-45 minutes.

In addition, the 7900HT system offers further high throughput options such as a 384-well block and a TaqMan Low Density Array Upgrade as well as a Zymark Twister Automation Accessory that will robotically load plates on the instrument.

For further information please speak with your local Life Technologies sales representative.

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Yes, you can run custom dyes, but the custom dye must fall into the wavelengths that are capable of being detected by a specific instrument. For optimal results on the filter-based instruments (including 7500 and 7500 Fast), the custom dye emission wavelength must fall into one of the emission filter peak detection wavelengths. For the laser-based 7900HT systems, the custom dye wavelength must fall within the overall range of detection for the instrument. Please refer to the Real-Time PCR information pages on our website under “Products & Services” for profiles of each instrument with a list of compatible standard dyes and the corresponding wavelengths supported. Please also refer to your User Manual or Installation and Maintenance Manual for instructions on how to calibrate custom dyes on your particular instrument.

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For computer and system requirements, please refer to the Release Notes for the ViiA 7 Software, which can be found on this page (http://www.thermofisher.com/us/en/home/technical-resources/software-downloads/applied-biosystems-viia-7-real-time-pcr-system.html )(Read Me).

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To open the software download on your Mac, you will need to adjust your security settings using the following instructions from Apple support:

Safely open apps on your mac

Please note, the software should run on Mac OS 10.10 or later.

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It is possible to import a standard curve into the Design & Analysis Software (v2.x) if you are analyzing results from one of the following Applied Biosystems real-time PCR instruments:
• 7900HT Fast Real-Time PCR System
• 7500/7500 Fast Real-Time PCR System
• StepOne/StepOne Plus Real-Time PCR System
• ViiA 7 Real Time PCR System
• QuantStudio 1 Real-Time PCR System
• QuantStudio 3 & 5 Real-Time PCR System
• QuantStudio 6 & 7 Flex Real-Time PCR System
• QuantStudio 6 & 7 Pro Real-Time PCR Systems
• QuantStudio 12K Flex Real-Time PCR system

Follow the instructions below to import a standard curve for analysis in the Design & Analysis Software (v2.x):
1. Open the data file in the Design & Analysis Software (v2.x).
2. Click on Actions.
3. Click on "Standard Curve Analysis Setting".
4. Select "External Standard Curves" and click on "Import".
5. Browse to the proper .csv file and click "Open".
6. Click "Apply".

If you are analyzing results from the ViiA 7 Real-Time PCR System, QuantStudio 6 Flex Real-Time PCR System, or QuantStudio 7 Flex Real-Time PCR System, it is possible to import a standard curve into the QuantStudio Real-Time PCR Software following the instructions below:
1. Open the data file in the QuantStudio Real-Time PCR Software.
2. Go to Analysis Settings.
3. Click on the "Standard Curve Settings" tab.
4. Select the option to "Use a standard curve imported from another experiment" and click on "Import".
5. Browse to and highlight the proper data file and click "Select".
6. Click Apply Analysis Settings.

No, the ViiA7 Real-Time PCR System is not compatible with the 7900HT Fast System Twister robot. For automation, the ViiA7 Real-Time PCR System can be connected to one of the following robots:
• Orbitor RS2 Microplate Mover for QuantStudio Systems, Sequential Access (cat. No. A32268)
• Orbitor RS2 Microplate Mover for QuantStudio Systems, Random Access (cat. No. A32269)
• Twister Automation Robot for QuantStudio Systems (Cat. No. 4471066)

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ViiA7 Software v1.2.4 was the last version of the ViiA7 Software released. Currently, the ViiA7 Real-Time PCR System can be controlled using the QuantStudio Software. To obtain the most recent copy of the QuantStudio Software for performing experiments on the ViiA7 Real-Time PCR System, please visit the instrument software download page, https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/applied-biosystems-viia-7-real-time-pcr-system.html.

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You can change the lamp in the ViiA7 Real-Time PCR System without the help of an engineer. Please follow the instructions provided in Appendix B of the following maintenance guide, https://tools.thermofisher.com/content/sfs/manuals/4489821.pdf. Once the lamp has been changed, you will need to calibrate the instrument again before performing a run.

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If you are running the QuantStudio Software v1.5.1 or below or the ViiA7 Software on the ViiA 7 Real-Time PCR instrument and using:
• A 384-well block: You may reach out to our Specialty Oligos team at Specialty_Oligos@thermofisher.com to request Cy3 dye or Cy5 dye calibration oligos. Each tube is provided at 800 µL, 0.2 µM. For a 384-well plate at 10 µL/well, you will need to order two tubes, or at 20 µL/well, you will need to order three tubes. Please provide your shipping address to Specialty Oligos and they will provide you with a formal quote.
• A 96-well block: You can use the Cy3 dye and Cy5 dye plate contained in either the 7500 Real Time PCR Systems Spectral Calibration Kit II (Cat. No. 4351151) (Standard, 0.2 mL) or the 7500 Fast Real-Time PCR Systems Spectral Calibration Kit II (Cat. No. 4362201) (Fast, 0.1 mL), depending on which 96-well block you have.

If you are running QuantStudio Software v1.6 or above on the ViiA 7 Real-Time PCR System and calibrating for:
• Cy5 dye: The spectral dye calibration plates for this software version include Cy5 dye and can be purchased online for the following block formats:
- 384-well block, https://www.thermofisher.com/order/catalog/product/A26335
- 96-well block, standard (0.2 mL), https://www.thermofisher.com/order/catalog/product/A26333
- 96-well block, Fast (0.1 mL), https://www.thermofisher.com/order/catalog/product/A26340
• Cy3 dye: You will have to follow the instructions shown above for QuantStudio software v1.5.1 and below and contact our Specialty Oligos team at Specialty_Oligos@thermofisher.com to request Cy3 dye calibration oligos.

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No, you do not need to back up the calibration files as all of the calibration files are stored on the ViiA7 Real-Time PCR instrument. Once the computer software is properly installed and the computer is connected to the instrument, the calibration information will automatically update in the QuantStudio Software.

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Yes, you can still start a run on the ViiA7 Real-Time PCR System if the expired calibration notice displays. This is just a reminder and you can close it to proceed with the run. Additionally, you can change when you want the notifications to show up and how frequently you want to be notified to calibrate the instrument. To do this, go to the toolbar in the QuantStudio Software and select Instrument > Instrument Console. Select your instrument icon and click on Manager Instrument > Maintenance > Calibration Reminders. From the Calibration reminders Settings page you can update when the calibrations expire, turn expiration reminders on/off, and set when the reminders show up.

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There are two main differences between a Fast 96-well and standard 96-well block for the Applied Biosystems real-time PCR systems:
• Reaction well volume: The Fast 96-well blocks have 0.1 mL reaction wells and are compatible with 0.1 mL plates and tubes. The standard 96-well blocks have 0.2 mL reaction wells and are compatible with 0.2 mL plates and tubes.
• Ramp rate: The fast 96-well blocks have a higher maximum ramp rate than standard blocks for the same real-time PCR system. However, both the fast 96-well and standard 96-well blocks are capable of running standard and fast chemistry.

Note: For newer instruments (QuantStudio 3 and 5 Real-Time PCR Systems and later) "0.1 mL" and "0.2 mL" are being used in place of "fast" and "standard" as designations for the two 96-well block formats.

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Yes, the 384-well block and TaqMan Array Card block use the same heated cover. However, each heated cover should only be used with the block it originally comes with. Pairing the cover with a different block may generate an instrument error message.

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All of the blocks on the ViiA 7 Real-Time PCR System can run fast cycling conditions. The ramp rates will vary between blocks.

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The following block types can be used on the ViiA 7 Real-Time PCR System:
• 96-well block
• Fast 96-well block
• 384-well block
• TaqMan Array Card block

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If you are running software v1.5.1 and below on the ViiA 7 Real-Time PCR instrument and using a 384-well plate, you may reach out to our Specialty Oligos team at Specialty_Oligos@thermofisher.com to request Cy5 dye calibration oligos. Each tube is provided at 800 µL, 0.2 µM. For a 384-well plate at 10 µL/well, you will need to order two tubes, or at 20 µL/well, you will need to order three tubes. Please provide your shipping address to Specialty Oligos and they will provide you with a formal quote.

If you are using a 96-well block, the Cy5 dye plate contained in either 7500 Real Time PCR Systems Spectral Calibration Kit II (Cat. No. 4351151) (Standard, 0.2 mL) or 7500 Fast Real-Time PCR Systems Spectral Calibration Kit II (Cat. No. 4362201 (Fast, 0.1 mL) can be used in place of the Cy5 dye calibration oligos.

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All Applied Biosystems instrument systems are qualified to detect a two-fold change. Scientifically speaking, there is a statistical calculation that predicts the probability of detecting one molecule in any given sample called the Poisson Distribution. The Poisson Distribution predicts that one molecule will be detected 66% of the time. This is assuming that the assay has been optimized to the fullest capacity and that there are no aliquoting or pipetting errors involved. Please refer to the following paper concerning one-copy detection: Lockey, C., E. Otto, and Z. Long. "Real-time fluorescence detection of a single DNA molecule." BioTechniques 24 (1998): 744-746.

Each assay designed on one of our platforms needs to be optimized to determine the Linear Dynamic Range of the assay. An Applied Biosystems Sequence Detection System (Real-Time PCR instrument) may be able to detect one copy in a fully optimized assay. As stated above, one copy can only be detected 66% of the time in a fully optimized system.

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Tutorials demonstrating introductions to Real-Time PCR, guidelines for designing real-time assays using Primer Express software, and various instrument set-ups can be found under Life Technologies University on our website. User bulletins, manuals and Product inserts, protocols can be found as downloadable pdf files on our Technical Resources page.

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All our real-time PCR instruments are compatible with Thermo Fisher Cloud Apps except the 7500 Fast instrument where the eds files alone are compatible with Thermo Fisher Cloud Apps.

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Please ask your dye manufacturer for recommendations. If there is no specific recommendation available, try TE Buffer (pH 8.0) to start.

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