TITLE: Qubit 4 Fluorometer (Common Peanut Lab)
AUTHORS: Nimalka Weerasuriya

[DESCRIPTION]
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.


[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.


[GUIDELINES]
Please ensure that all reagents have been stored appropriately and are not expired before running this protocol. 

[STEPS]
SECTION: Introduction
2. 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.
SECTION: Reagent Calculator (Optional)
3. Skip this section if the kit comes with a bottle of Working Solution, or if you are using the Protein Broad Range Assay.
 

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. 

On the Home screen, press Reagent Calculator.
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.


SECTION: Reagent Calculator (Optional)
4. Prepare the Qubit Working Solution with the shown inputs.
 

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.

SECTION: Introduction
1. 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.
SECTION: Troubleshooting
15. Calibration error message:
 
In the Error screen, press OK.
Review the Read standard screen.
If you wish to re-run the standards  , or run new standards   , press Read standards, then repeat the calibration procedure.
SECTION: Read Samples
7. Before you begin:
Make sure you have either run new standards   or accepted values from the previous calibration.
Prepare the samples. Refer to   

 

SECTION: Calibrating New Standards (Optional)
6.4. 
 
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.
 

If you receive a "Calibration error" message, see   
SECTION: Calibrating New Standards (Optional)
6.3. At the prompt, insert the prepared    into the sample chamber and press Read standard. 
For Qubit protein and RNA IQ assays only: At the prompt, insert Standard #3 into the sample chamber and press Read standard.
The calibration is complete and the software displays the results.
SECTION: Calibrating New Standards (Optional)
6.2. Prior to running samples
At the prompt, insert the prepared   into the sample chamber and press Read standard. 


SECTION: Calibrating New Standards (Optional)
6.1. 
 
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.
SECTION: Calibrating New Standards (Optional)
6. 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
SECTION: Read Samples
8. Press Run samples.
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.

 

SECTION: Read Samples
8.1. 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.

 

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

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).
SECTION: Results
11. 
 
If the results are outside of the assay's range, an "Out of Range" message is displayed. See   for adjustments.

SECTION: Results
10. To view the Fluorescence vs. Concentration graph, swipe left or press the right arrow. In the 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.
SECTION: Other Modes
14. 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)
SECTION: Managing Data
12. 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).
SECTION: Managing Data
13. Sample details can be seen by going to the Home screen, pressing Data.
On the Export data screen, press the data set of interest. 
A Data screen displaying a list of data entries for that set appears. Scroll to view additional entries.
To view sample details, press the sample of interest. A Data details screen opens.
 

SECTION: Cleaning &amp; Updates
21. 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.
SECTION: Cleaning &amp; Updates
22. Software updates can be done by downloading the latest software to a USB drive from thermofisher.com/qubit.
If using a USB, insert into instrument.
On the Home screen, press Settings.
Press Update software. The instrument will search the USB drive. 
When update files are detected, press Update to update the software.
When prompted, Restart to complete the update.
SECTION: Troubleshooting
20. 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.
SECTION: Troubleshooting
16. 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.
SECTION: Troubleshooting
17. Reading Out of Range (High):
 

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.
SECTION: Troubleshooting
18. Reading Out of Range (Low):
 

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.
SECTION: Troubleshooting
19. 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. 
SECTION: Preparing Samples
5. 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:
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. 
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.
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).
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. 
SECTION: Preparing Samples
5.1. Set up the required number of 0.5-mL tubes for standards (optional) and samples. Each assay requires 2 standards.
Label the tube lids.

 

SECTION: Preparing Samples
5.2. 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.
 

SECTION: Preparing Samples
5.3. (Optional) Standards:
Add 190 µL of working solution to each of the tubes used for standards. 
Add 10 µL of each standard to the appropriate tube, then mix by vortexing 2–3 seconds. Be careful not to create bubbles. 
 

SECTION: Preparing Samples
5.4. Samples:
Add working solution to individual assay tubes so that the final volume in each tube after adding sample is 200 µL. 
 

SECTION: Preparing Samples
5.5. 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 200 µL Read Samples. 
 

Allow all tubes to incubate at Room temperature for 2 min.
SECTION: Preparing Samples
None. The kits include concentrated assay reagent, dilution buffer [or pre-mixed working solution] and 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), then read the concentration using the Qubit Fluorometer. 
The assay is highly selective for double-stranded DNA (dsDNA) over RNA and is accurate for initial sample concentrations from 10 pg/µL to 100 ng/µL. 
The assay is performed at room temperature, and the signal is stable for 3 hours. 
Common contaminants such as salts, free nucleotides, solvents, detergents, or protein are well tolerated in the assay.
SECTION: Preparing Samples
None. Set up the required number of 0.5-mL tubes for standards and samples. The dsDNA HS Assay requires 2 standards.
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.
SECTION: Preparing Samples
None. Optional: Prepare the Qubit Working Solution (if it is not already provided) by diluting the dsDNA HS Reagent 1:200 in dsDNA HS Buffer. Use a clean plastic tube each time you prepare working solution. Do not mix the working solution in a glass container.
 

SECTION: Preparing Samples
None. Standards:
Add 190 µL of working solution to each of the tubes used for standards. 
Add 10 µL of each standard to the appropriate tube, then mix by vortexing 2–3 seconds. Be careful not to create bubbles. 
 

SECTION: Preparing Samples
None. Samples:
Add working solution to individual assay tubes so that the final volume in each tube after adding sample is 200 µL. 
 

SECTION: Preparing Samples
None. 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 200 µL Read Samples. 
 

Allow all tubes to incubate at Room temperature for 2 min. Then  .
SECTION: Preparing Samples
None. Overview

The kits include concentrated assay reagent, dilution buffer [or pre-mixed working solution] and prediluted DNA standards. 
If there is no pre-mixed working solution, simply dilute the RNA BR reagent 1:200 in BR buffer provided, add your sample (any volume from 1–20 μL is acceptable), then read the concentration using the Qubit Fluorometer. 
The assay is highly selective for RNA over double-stranded DNA (dsDNA). Depending on sample volume, the assay is accurate for initial sample concentrations from 0.5 ng/μL to 1200 ng/μL.
Common contaminants such as salts, free nucleotides, solvents, detergents, or protein are well tolerated in the assay.
The RNA BR assay is intended for total RNA, rRNA, or large mRNA. For small RNA (∼20 nt or bp), we recommend the Qubit microRNA Assay Kit (Cat. Nos. Q32880, Q32881).

We highly recommend treating the rRNA standards as you would any other RNA, including:
Use appropriate RNAse-free handling techniques, including RNAse-free gloves, pipette tips, and tubes.
Keep the tube lids closed whenever possible.
Do not touch the pipette to the inside wall of the tube when withdrawing a sample.
Return the rRNA standard to the refrigerator as soon as possible after use
SECTION: Preparing Samples
None. Set up the required number of 0.5-mL tubes for standards and samples. The RNA BR Assay requires 2 standards.
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.
SECTION: Preparing Samples
None. Optional: Prepare the Qubit Working Solution (if it is not already provided) by diluting the RNA BR Reagent 1:200 in RNA Buffer. Use a clean plastic tube each time you prepare working solution. Do not mix the working solution in a glass container.
 

SECTION: Preparing Samples
None. Standards:
Add 190 µL of working solution to each of the tubes used for standards. 
Add 10 µL of each standard to the appropriate tube, then mix by vortexing 2–3 seconds. Be careful not to create bubbles. 
 

SECTION: Preparing Samples
None. Samples:
Add working solution to individual assay tubes so that the final volume in each tube after adding sample is 200 µL. 
 

SECTION: Preparing Samples
None. Add each sample to the assay tubes containing the correct volume of working solution, then mix by vortexing 3-5 seconds. The final volume in each tube should be 200 µL Read Samples. 
 

Allow all tubes to incubate at Room temperature for 2 min. Then  .
SECTION: Preparing Samples
None.