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How do I start a Q96 experiment and calculate cells per sample?

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Overview:

This guide provides comprehensive instructions on how to initiate an experiment using the LuminUltra GeneCount Q96 fluorescence quantitative PCR system and calculate cells per sample once your experiment is complete. Follow these steps to set up your experiment, run it, and analyze your results effectively.

Part 1: Starting a Q96 Experiment

1. Powering On the System:

    • Check the Environment: Ensure the instrument is placed in a well-ventilated, low-humidity area and connected to a grounded power outlet.
    • Power Up: Turn on the system by flipping the power switch on the back panel of the Q96.
    • Launch the Software: Open the GeneCount Q96 software on your computer or tablet and confirm it connects to the device.

2. Logging In and Selecting an Experiment Template:

      • Login with Credentials: Use your account information to log into the software.
      • Load a Template: Select a template suitable for your assay (e.g., pathogen detection, DNA quantification). Templates contain pre-set detectors and cycling parameters, which streamline setup and reduce error. If no templates are visible, visit GeneCount® Q-96 Software Experiment Templates
      • Add Experiment Details: Enter the experiment name, user information, and relevant notes in the “Detector” tab for easy future reference. Remember to save your experiment. 

3. Configuring Sample Information:

    • Navigate to Sample Setup: Click Setup > Sample to begin entering sample data.
    • Add Samples:
      • Single Entry: Enter a Sample ID manually and hit Enter. 
      • Batch Entry: Use the “Batch Add” function to input multiple samples. Input the starting Sample ID and number of samples, then click Add.
    • Double click to bring up colour palette and change the colour of each sample. 
    • Verify Sample Details: Double-check that all sample IDs and details are correctly entered to avoid errors during analysis.

4. Setting Up the Reaction Plate:

    • Open Reaction Plate Layout: In the software, navigate to Setup > Plate to access the reaction plate layout. This layout represents each well on the 96-well plate, allowing you to assign sample types and specify each well’s contents.
    • Arrange Samples in the Plate:
      • Manual Placement: Select each well individually and assign samples by clicking the corresponding sample type and property. Double-check well assignments to avoid errors.
      • Auto Arrange Feature: For larger data sets, use the Sample Auto Arrange feature, which automatically fills selected wells based on your sample list. This feature ensures a systematic and error-free placement, particularly useful for high-throughput experiments.

    • Assign Sample IDs and Properties:

      • Select each well on the plate layout, then assign the Target

    • Define Sample Types for Each Well:

      • Unknown (U): For test samples with unknown target concentrations. Each sample’s fluorescence data will be recorded, allowing for quantitative analysis.
      • Positive Control (P): Contains a known concentration of the target material to confirm the assay’s accuracy and ensure reliable detection.
      • Negative Control (N): This well contains only the master mix and nuclease-free water, with no target DNA/RNA. Negative controls help verify that there is no contamination, which could lead to false positives.
    • Loading Samples: Insert your tubes or 96-well plate into the instrument’s heat block, following the on-screen layout.

5. Starting the Experiment:

    • Save Experiment Setup: Before beginning, save your experiment configuration using the “Save As” option.
    • Begin the Run: Go to the “Run” tab, click “Start Run,” and monitor real-time fluorescence data on the “Fluorescence Curve” tab. The system will beep when the run completes.

Part 2: Calculating Cells per Sample from the Q96 Results

  1. View Results in the Analysis Page:

    • Once your Q96 experiment is complete, navigate to the Analysis page within the software. Here, you’ll find all recorded data, including the cycle threshold (Ct) values and fluorescence curves for each well.

  2. Exporting the Experiment Data:

    • Click Export Experiment in the software, then select Export to Excel and click OK to confirm. This will generate an Excel file containing your experimental data.
    • Transfer Option: If you are working directly on the Q96 interface, you can transfer this exported file to your PC for easier analysis.

  3. Open the Exported Data File:

    • Locate the downloaded Excel file on your computer. This file includes essential information like Ct values that you will need for further calculations in the quantification workbook.

  4. Open the Q96 Quantification Workbook:

    • Launch the Q96 Quantification Workbook on your computer. This workbook is designed to streamline data input and calculations for determining sample concentrations.
    • Note: Follow the instructions listed in the workbook, especially step 5, which directs you to locate and use data from the Quan Results tab.

  5. Copy Data from the Quan Results Tab:

    • In the Excel file exported from the Q96, navigate to the Quan Results tab. Select and copy all results from this tab, then paste them into the designated area in the Q96 Quantification Workbook.

  6. Input Additional Sample Information:

    • Depending on your sample type, enter the necessary values into the quantification workbook to complete the calculation:
      • Sample Volume: For liquid samples.
      • Swab Area: For surface swab samples.
      • Sample Weight: For solid or particulate samples.
    • These inputs ensure accurate concentration calculations tailored to your sample’s characteristics.

Note: If any of the controls fail or if you encounter issues during these steps, please refer to our Help Center for troubleshooting articles or raise a support ticket for additional assistance.

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