Feb 21, 2024

Public workspaceQubit 4 Fluorometer (Common Peanut Lab)

This protocol is a draft, published without a DOI.
  • 1USDA, Oklahoma State University, Stillwater
Nimalka Weerasuriya
Open access
Protocol Citation: Nimalka Weerasuriya 2024. Qubit 4 Fluorometer (Common Peanut Lab). protocols.io https://protocols.io/view/qubit-4-fluorometer-common-peanut-lab-c78ezrte
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 25, 2024
Last Modified: February 21, 2024
Protocol Integer ID: 94182
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This protocol is to guide the use of the Qubit 4 Fluorometer in the Stillwater, OK, ARS USDA facility. The complete user guide is also available for download at thermofisher.com/qubit. This guide contains some additional information for Common Lab use and may not be applicable for everyone.

The Qubit 4 Fluorometer is a benchtop fluorometer that can be used for the quantitation of DNA, RNA, microRNA, and protein, as well as for the measurement of RNA integrity and quality using the highly sensitive and accurate fluorescence based Qubit assays. You can also use the Qubit 4 Fluorometer to directly measure the fluorescence of samples or to create new assays using the MyQubit software preprogrammed into the instrument.

Guidelines
Please ensure that all reagents have been stored appropriately and are not expired before running this protocol.
Materials
Assay Tubes for the Qubit 4:
Only thin-wall, clear 0.5 mL PCR tubes are appropriate for use in the Qubit 4. Acceptable tubes include the Qubit assay tubes (Cat. No. Q32856, 500 tubes). The minimum assay volume must be 200 µL for an accurate read.

To Perform Assays:
  • A Qubit assay kit appropriate for quantifying your samples (see page 61 of the full manual for available Qubit kits and ordering information)
  • DNA, RNA, or protein samples
  • Qubit assay tubes or other appropriate 0.5 mL assay tubes
  • Appropriate standards for the assay
  • Optional: USE drive or USB cable for data transfer


Before start
To power up the Quibit, plug the power cord into the electrical outlet. It will power on and first display the splash screen, and then the Home screen.

Introduction
Introduction
General Guidelines:
  • Wear gloves during all sample handling.
  • Bring all kit reagents to room temperature and insert all assay tubes into instrument only for as much time as it takes for the instrument to measure the sample.
  • Do not hold the assay tubes in your hand before performing the measurement.
  • Incubate the DNA and RNA assay tubes for 2 minutes after mixing the sample or standard with the working solution.
  • Incubate the protein assay tubes for 15 minutes after mixing the sample or standard with the working solution.
  • Incubate the Broad Range Protein assay tubes for 10 minutes after mixing the sample or standard with the Protein BR Assay Reagent.
  • If you are performing multiple readings of a single tube, remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading.
Note: multiple readings of RNA samples is not recommended.
From the Home screen, you can:
  • Select one of the assays: dsDNA, RNA, oligo (ssDNA), protein, or Ion Sphere™.
  • Select the Fluorometer mode (the instrument behaves like a mini-fluorometer).
  • Access and export saved data.
  • Configure instrument settings.
  • Use the Reagent Calculator to determine the exact volumes of Qubit™ buffer and reagent required to prepare the Qubit™ working solution.
Reagent Calculator (Optional)
Reagent Calculator (Optional)
Skip this section if the kit comes with a bottle of Working Solution, or if you are using the Protein Broad Range Assay.
Enter number of samples
and standards required to
run assays.

Use the on-board Reagent Calculator to quickly determine the correct amount of Qubit dye and buffer required to make the appropriate amount of Working Solution to prepare samples and standards.

  1. On the Home screen, press Reagent Calculator.
  2. Enter the total number of samples and standards that you will be running. Press Enter.

Optional: Select Include Overage if you want to include an additional tube in your calculations in case of pipetting error.


Prepare the Qubit Working Solution with the shown inputs.
Prepare your Working Solution
as shown above in a separate
sterile tube.

Note. You can change the total number of tubes that you plan to run or the overage selection on this screen as well.

Press Done to return to the Home screen to run the assay.

Preparing Samples
Preparing Samples
2m
Multiple kits can be used for one sample to determine purity:
  • Use the dsDNA BR Assay Kit together with the RNA BR Assay Kit. These measurements give you a better indication of sample purity than that produced by measuring the A260/A280 ratio. To measure protein contamination in nucleic acid samples, simply run 1–20 µL of the sample in the Qubit Protein Assay.

Kit Options:
  1. dsDNA Broad Range Kit: The assay is highly selective for double-stranded DNA (dsDNA) over RNA and is accurate for initial sample concentrations from 100 pg/µL to 1000 ng/µL.
  2. dsDNA High Sensitivity Kit: The assay is highly selective for double stranded DNA (dsDNA) over RNA. Depending on sample volume, the assay is accurate for initial sample concentrations from 5 pg/µL to 120 ng/µL providing an assay range of 0.1–120 ng.
  3. RNA BR Kit: This assay is highly selective for RNA over double-stranded DNA (dsDNA) and is accurate for initial sample concentrations from 0.5 ng/μL to 1200 ng/μL, providing an assay range of 10−1,200 ng. The RNA BR assay is intended for total RNA, rRNA, or large mRNA. For small RNA (∼20 nt or bp), we recommend the microRNA Assay Kit (Cat. Nos. Q32880, Q32881).
  4. Protein BR Kit: provides a quick and accurate method to quantitate protein samples over a broad range of concentrations for sample concentrations from 100 µg/mL to 20 mg/mL.

The kits include concentrated assay Reagent, dilution Buffer [or pre-mixed working solution] and two prediluted DNA standards.
  • If there is no pre-mixed working solution, simply dilute the reagent using the buffer provided, add your sample (any volume from 1–20 μL is acceptable).
  • The assays are performed at room temperature, and the signal is stable for 3 hours.
  1. Set up the required number of 0.5-mL tubes for standards (optional) and samples. Each assay requires 2 standards.
  2. Label the tube lids.

Note
Do not label the side of the tube as this could interfere with the sample read. Label the lid of each standard tube correctly. Calibration of the Qubit requires the standards to be inserted into the instrument in the right order.

Optional: Prepare the Qubit Working Solution (if it is not already provided) by diluting the kit's Reagent 1:200 in the kit's Buffer solution. Use a clean plastic tube each time you prepare working solution. Do not mix the working solution in a glass container.
Note
The final volume in each tube must be 200 µL. Each standard tube requires 190 µL of working solution, and each sample tube requires anywhere from 180–199 µL. Prepare sufficient working solution to accommodate all standards and samples.

For example, for 8 samples, prepare enough working solution for the samples and 2 standards: ~200 µL per tube in 10 tubes yields 2 mL of working solution (10 µL of Qubit reagent plus 1990 µL of Qubit buffer).

(Optional) Standards:
  1. Add Amount190 µL of working solution to each of the tubes used for standards.
  2. Add Amount10 µL of each standard to the appropriate tube, then mix by vortexing 2–3 seconds. Be careful not to create bubbles.
Note
Careful pipetting is critical to ensure that exactly 10 µL of each Qubit standard is added to 190 µL of working solution.

Samples:
  1. Add working solution to individual assay tubes so that the final volume in each tube after adding sample is 200 µL.
Note
Your sample can be anywhere from Amount1-20 µL . Add a corresponding volume of working solution to each assay tube: anywhere from Amount180-199 µL .

  1. Add each sample to the assay tubes containing the correct volume of working solution, then mix by vortexing 2–3 seconds. The final volume in each tube should be Amount200 µL .
Note
If you are adding 1–2 μL of sample, use a 2-μL pipette for best results.

  1. Allow all tubes to incubate at TemperatureRoom temperature for Duration00:02:00 minutes .
2m
Calibrating New Standards (Optional)
Calibrating New Standards (Optional)
For each assay, you have the choice to run new standards for calibrating the assay, or to use values from the previous calibration (for more information see page 60 in the manual).

On the Home screen, select the assay type. Swipe left or right to view all pages.
Note: The software displays the available assays for the assay type you selected.

Assay options:
  • dsDNA (genomic or PCR products): High sensitivity or Broad range
  • RNA Broad Range
  • Protein Broad Range


If you have already performed a calibration for the selected assay, the software prompts you to choose between reading a new standard set and running samples using the previous calibration. Press Read standards.

Note: To apply the previous calibration to your sample readings, press Run samples. The software prompts you to insert the assay tube containing the sample. See "Read Samples" section 8.
Prior to running samples
  1. At the prompt, insert the prepared SampleSample into the sample chamber and press Read standard.


  1. At the prompt, insert the prepared SampleSample into the sample chamber and press Read standard.
  2. For Qubit protein and RNA IQ assays only: At the prompt, insert Standard #3 into the sample chamber and press Read standard.
  3. The calibration is complete and the software displays the results.

Standard fluorescence vs. concentration graph.
For Qubit quantitation assays: If the calibration is successful, the software displays the Read standard screen with the Fluorescense vs. Concentration graph.

The standard data points are connected by a line.
  • For the RNA IQ assay: If the calibration is successful, the software displays the Ready for samples message.

If the calibration is not successful, see Troubleshooting section.
Note
The Read standard screen displays raw fluorescence values for Standard #1 and Standard #2 (and Standard #3 if applicable). These values can assist in making judgements regarding the calibration results.
  • For the dsDNA BR, dsDNA HS, ssDNA, microRNA, RNA HS, RNA BR, RNA XR and protein assays, the reading given by Standard #2 should be at least 10x higher than Standard #1.
  • For the protein assay, the reading given by Standard #3 should be at least 40% higher than that of Standard #2.

If you receive a "Calibration error" message, see Go togo to step #15 Troubleshooting
Read Samples
Read Samples
Before you begin:
  • Make sure you have either run new standards Go to Calibrating New Standards or accepted values from the previous calibration.
  • Prepare the samples. Refer to Go togo to step #5 Preparing Samples

Note
Incubate the samples for the appropriate amount of time after mixing them with the Working Solution (2 minutes for DNA and RNA assays, 15 minutes for protein, or 10 minutes for the Protein Broad Range Assay).

  1. Press Run samples.
  2. In the Sample volume screen, select the sample volume and units for the quantitation assays or the RNA IQ assay:
  • Press the + or - buttons to select the sample volume added to the tube (between 1 and 20 µL)
  • Select the units for the output sample concentration in the dropdown menu. Typical units are ng/uL for DNA assays.

Selecting sample volume and output units for quantitation assays.

Insert a sample tube into the chamber, close the lid and press Read tube. The reading takes 3 seconds for quantitation and 5 seconds for quality assays.

(L) Sample quantitation result; (R) RNA IQ result

To read multiple samples for the same assay:
  1. Remove the current sample, insert a new sample.
  2. To change the sample volume, swipe right or press the left arrow.
  3. Press Read tube.
  4. Repeat step 2 and 3.
Results
Results
The Results screen displays the results of the sample run.
Results after quantitation.

  • If the results are within the assay's range, the concentration values are displayed. The top value (in large font) is the concentration of the original sample. The bottom value is the dilution concentration (the concentration of the sample in the tube inserted into the fluorometer).
To view the Fluorescence vs. Concentration graph, swipe left or press the right arrow. In the graph:
Fluorescence vs Concentration graph.
  • Open circles represent the correct standards.
  • The large gray circle represents the most recent sample.
  • Blue circles represent samples that fall within the assay's core range.
  • Yellow circles represent samples that fall within the assay's extended range.
  • Red circles represent samples or standards that fall outside the assay's range.


If the results are outside of the assay's range, an "Out of Range" message is displayed. See Go togo to step #15 Troubleshooting for adjustments.

Managing Data
Managing Data
The Qubit can save data for up to 1000 samples. For the saved data, the instrument allows you to:
  • View detailed data for each sample (manual page 32).
  • Rename data files (manual page 35).
  • Save data as a CSV or PDF file, and export to a USB dive (page 36).
  • Delete data files (page 40).
  1. Sample details can be seen by going to the Home screen, pressing Data.
  2. On the Export data screen, press the data set of interest.
  3. A Data screen displaying a list of data entries for that set appears. Scroll to view additional entries.
  4. To view sample details, press the sample of interest. A Data details screen opens.


Other Modes
Other Modes
The Qubit can also be used as:
  • a mini-fluorometer using the Fluorometer mode (see pages 29-30 in manual).
  • Ion Sphere Assay for Ion Sphere Particles prior to a sequencing run on an Ion Personal Genome Machine Sequencer (see page 31)
Troubleshooting
Troubleshooting
Calibration error message:
Calibration error screen.
  1. In the Error screen, press OK.
  2. Review the Read standard screen.
  3. If you wish to re-run the standards Go togo to step #6.2 Running Standards , or run new standards Go to Preparing Standards , press Read standards, then repeat the calibration procedure.
Handling samples:
  • For RNA tests, use appropriate RNAase-free handling techniques, including RNAse-free gloves, pipette tips, and tubes. Keep the tube lids closed whenever possible; do not press the pipette to the inside wall of the tube when withdrawing a sample. Return the RNA standards to -80°C as soon as possible.
  • Ensure the assay tubes are at room temperature at time of reading. Do not hold them in your hand and do not leave them in the Fluorometer for longer than it needs to take a measurement.
  • Use clean 0.5 mL PCR tube for each reading. Tubes must be dry on the outside. Moisture and condensation will lead to reading errors.
  • Minute bubbles in samples will cause reading errors. Be sure not to introduce bubbles into samples. Slight tapping on the tube wall or brief centrifugation will often help dissipate bubbles.
Reading Out of Range (High):
Sample High Reading

  • The sample is out of range. Use a sample that is less concentrated (for example, 10 µL in 190 µL instead of 20 µL in 180 µL)
  • For quantitation assays, view the Fluorescence vs. Concentration graph in the Results screen to confirm that the values for the samples fall between the standards.
  • Ensure that the lid is closed when reading standards and samples.
  • Prepare samples and standards according to the assay kit you are using.
  • Ensure that the assay is performed entirely at room temperature.
Reading Out of Range (Low):
Sample Low Reading

  • Use a sample that is more concentrated or use a lower dilution (for example, 20 µL in 180 µL instead of 10 µL in 190 µL).
  • For quantitation assays, view the Fluorescence vs. Concentration graph in the Results screen to confirm that the values for the samples fall between the standards.
  • Ensure that you have prepared the Working Solution correctly (1:200 dilution using kit buffer).
  • Ensure that the standard sample tubes are filled to 200 µL.
  • Protect the reagent and working solutions from light.
  • Calibrate the standards again. Standards must be used in the correct order.
  • Ensure that the assay is performed entirely at room temperature.
Critical Considerations:
  • Allow for the appropriate incubation time.
  • Prevent photobleaching of reagents.
  • The temperature inside the Qubit may be as mush as 3°C above room temperature after 1 hour. If you want to perform multiple readings of a single tube, remove the tube and wait 30 seconds before re-inserting.
  • Assays were designed to be performed at 22-28°C and temperature fluctuations can influence the accuracy of the assay. Store all kit reagents at room temperature and do not hold tubes in your hand before a measurement.
  • For each assay you can run a new calibration or use the values from previous calibrations. Calibration data may be affected by pipetting accuracy and temperature fluctuations.
System verification test:
The system verification test checks the internal components and requires the use of the System Verification Assay Kit (Cat. no. Q33237). Perform the system verification when a problem with the instrument is suspected. It is not necessary to perform this regularly. See pages 55-56 in the manual.
Cleaning & Updates
Cleaning & Updates
The Qubit does not require regular maintenance.

To clean the fluorometer periodically:
  • the touchscreen can be cleaned after disconnecting the power cable, using a soft cloth lightly moistened with LCD cleansing detergent.
  • To disinfect, disconnect the power, and clean using a soft cloth lightly moistened with 70% ethanol, 70% isopropanol or 10% bleach.
  • The cloth included with the instrument is only recommended for use with LCD detergent.
Software updates can be done by downloading the latest software to a USB drive from thermofisher.com/qubit.
  1. If using a USB, insert into instrument.
  2. On the Home screen, press Settings.
  3. Press Update software. The instrument will search the USB drive.
  4. When update files are detected, press Update to update the software.
  5. When prompted, Restart to complete the update.