Nucleic Acid Quantification Support—Troubleshooting

There are several reasons why this message may appear. Please check the following on your instrument:

1. View the raw fluorescence value for the standards under “Check Standards” or "Check Calibration." Is the value of Standard 2 at least 10- to 50-fold higher than the value of Standard 1? For the protein assay, is the value of Standard 3 about twice the value of Standard 2? Check to see that you have used the tubes in the correct order.
   
2. Check to see that you used 10 µL of the standard and 190 µL of the Qubit® dye working solution in the appropriate tubes.
   
3. For RNA assays, try an unopened tube of Standard 2 in case the open RNA standard tube has degraded.
   
4. Check the age of the Qubit® assay kit and standards. Kits are stable for at least 6 months from the date of receipt. If the kit is older than a year and the Qubit® Fluorometer will not read the standards, it may need to be replaced with a new kit.
   
5. Check to see if the instrument is working properly. Lift the lid. Run a new calibration for any assay. You should see a red light (for RNA assays) or a blue light (for all other assays) flash 5 times.

If this occurs, view the raw fluorescence values for the standards and samples under “Check Standards” or "Check Calibration." Confirm that the values for the samples fall between the values of the standards (or a little above the highest standard). If they do not, the sample is out of the accurate range of the assay. The Qubit® Fluorometer screen should indicate OUT OF RANGE.

Dilute your sample, or use a smaller volume of sample in the assay and try again. Alternatively, if you are using the Qubit® DNA HS Assay or the Qubit® RNA HS Assay, try using the Qubit® DNA BR Assay or Qubit® RNA BR Assay, as these have lower sensitivity.

You can try to use a larger amount of sample if possible (up to 20 µL). Alternatively, if you are using the Qubit® DNA BR Assay or Qubit® RNA BR Assay, try using the Qubit® DNA HS Assay or Qubit® RNA HS Assay, as these have higher sensitivity.

Most likely, the sample is contaminated with other molecules that absorb at 260 nm. The NanoDrop® Spectrophotometer is reading these contaminants, but the Qubit® Fluorometer is not. To determine the exact composition of the sample, perform a Qubit® dsDNA Assay, Qubit® RNA HS Assay, and Qubit® Protein Assay on small aliquots of the same sample. Check the Qubit® assay kit product manual for a list of contaminants that could interfere with the assay. Dilute or purify the sample further to reduce contaminant concentration. Mix the working solution and the sample aliquoted into it well.

Please see our suggestions below:

• Make sure that you take your reading only after incubating for 2 minutes (15 minutes for protein).
• If you leave the assay tube in the Qubit® Fluorometer and take multiple readings, the readings will go down as the tube heats up inside the instrument. If you want to take multiple readings, remove the tube from the instrument, place it in a tube rack, and allow it to equilibrate to room temperature for at least 30 seconds before rereading the tube.
• You may read the sample up to 3 hours after mixing if it is protected from light. After this time, the reading will not be accurate.
• Keep standards and sample tubes in the dark and protected from light in between readings.

Please see the possibilities below:

• If you have been reading UV absorbance and your value is lower than expected, it is likely that your sample is contaminated with other molecules that absorb at 260 nm.
• The initial value that the Qubit® Fluorometer shows is the concentration of the biomolecule (DNA, RNA, or protein) in the assay tube. To determine the concentration of the biomolecule in the SAMPLE, use the "Calculate Stock Concentration" or "Calculate Sample Concentration" feature on the results screen.
• Check the Qubit® assay kit product manual for a list of contaminants that could interfere with the assay.
• Ensure that the lid is completely closed when reading standards and samples.
• Ensure that you have made a fresh dilution of the dye in buffer (1 µL dye to 199 µL buffer.)
• Ensure that you have labeled your tubes correctly.
• Ensure that the kit has not passed its expiration date.
• Ensure that the dye has been stored in the dark.
• Ensure that the buffer and dye are both stored at room temperature. It takes hours for a bottle of buffer at 4°C to warm to room temperature.
• Check for air bubbles in the solution. Bubbles on top are OK, but bubbles lower in the solution or at the bottom may affect the reading.
• To reduce pipetting error, dilute the sample and use a larger volume. This is especially important when pipetting concentrated or viscous solutions. See graph below.

Standards for the 2 μL aliquots were made from 500 µg/mL lambda phage DNA stock diluted to 25, 50, 100, 300, 400, and 500 µg/mL. Standards for the 10 μL aliquots were 5X dilutions of the 2 μL standards. Standards for the 20 μL aliquots were 2X dilutions of the 10 μL standards. Note that using 2 µL of the highest concentrations of lambda DNA standards gave lower than expected fluorescence in the assay.

Here are several suggestions:

1. View the raw fluorescence value (RFU) for the standards under “Check Standards” or "Check Calibration." Confirm that the values for the samples fall between the values of the standards (or a little above the highest standard). If they do not, the sample is out of the accurate range of the assay. Refer to the confidence ranges for each assay in the product manuals. The readout in the assay will be to 2 significant figures instead of 3 if the assay sample is out of the high confidence range.
   To bring the sample into the accurate range, dilute the sample or use more or less of it (for example, 10 µL instead of 2 µL if the sample reads low).
   
2. Check for temperature issues: The assay is temperature sensitive and the fluorescent signal can decrease at higher temperatures. Temperature fluctuations between samples, or between samples and standards, can cause problems.
   Make sure that the buffer and Qubit® reagent in DMSO are at room temperature. The buffer and Qubit® reagent should be stored at room temperature, not in the refrigerator. Even after 2–3 hours at room temperature, buffer previously stored at 4°C can remain below room temperature.
   Make sure your samples and working solution are not too warm (including those straight from a centrifuge). Samples kept in the Qubit® instrument too long or read multiple times can warm up. If you want to perform multiple readings of a single tube, you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading. Also, do not hold tubes in your hand for very long before reading them in the instrument, since this can warm the sample, resulting in a low reading.
   
3. Ensure that you have prepared the Qubit® working solution correctly (1:200 dilution using the buffer provided in the kit). Ensure that you have prepared the standard tubes correctly (10 µL of each standard in 190 µL of the working solution). Ensure that the tubes are filled with at least 200 µL (both standards and samples).
   
4. Ensure that the reagents and standards you are using are less than 1 year old, and that the standards have been stored correctly. The Qubit® reagent stock solution should be protected from light as much as possible.
   
5. Ensure that you have selected the correct assay on the Qubit® Fluorometer for the Qubit® assay you are performing.
   
6. Ensure that the lid is completely closed when reading standards and samples.
   
7. Use recommended tubes (both so the tube does not obstruct the lid, and for optical clarity). Some types of tubes can have high autofluorescence that will affect the assay.
   
8. Did you enter the number of microliters of stock you pipetted into the working solution into the Qubit® instrument? If so, the reading after giving the Qubit® Fluorometer this information is the concentration of your stock solution. If you did not, the reading you got is for the concentration in the assay tube (the tube you put into the Qubit® Fluorometer)—not your stock solution.
9. If you are comparing Qubit® assay results to concentration obtained by UV absorbance, and the concentration based on absorbance is significantly higher, it may be because of nucleic acid or protein contamination. The Qubit® assays are much more specific for DNA, RNA, or protein than absorbance readings.

Please download and install the most current version of the Qubit® Firmware from our website.
For a Qubit® 2.0 Fluorometer with a serial number below 1104004846, please use a Windows® operating system to update your Qubit® 2.0 Fluorometer using the instructions on this page. If you do not have access to a PC, please contact Technical Support at techsupport@lifetech.com for further instructions.
Click on the appropriate link below to install the needed firmware update:
Qubit® 2.0 Firmware Update
Qubit® 1.0 Firmware Update (see bottom right-hand corner of the page)

Absorbance readers cannot distinguish small fragments, degraded samples, or single nucleotides from intact nucleic acid. The dyes in the kits only detect intact strands longer than about a 20-mer. The kits are only recognizing intact DNA or RNA, and ignoring degraded sample.

• The sample is out of range. Use a sample that is less concentrated.
• View the Fluorescence vs. Concentration graph in the Results screen to confirm that the values for the samples fall between the values of the standards (see page 24 of the manual).
•  Ensure that the lid is closed while reading standards and samples.
•  Prepare samples and standards according to the instructions in the Qubit® assay kit you are using.
• Ensure that the assay is performed entirely at room temperature.

• The sample is out of range. 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).
• View the Fluorescence vs. Concentration graph in the Results screen to confirm that the values for the samples fall between the values of the standards (see page 24 of the manual).
• Ensure that you have prepared the Qubit® working solution correctly (1:200 dilution using the buffer provided in the kit).
• Ensure that you have prepared the standard tubes correctly (10 μL of each standard in 190 μL of Qubit® working solution).
• Ensure that the standard and sample tubes are filled to 200 μL.
•  Protect the Qubit® reagent and working solutions from light.
• Select the correct Qubit® 3.0 Fluorometer assay for the Qubit® assay you are performing and calibrate the fluorometer correctly. Standards must be used in the correct order.
• Ensure that the assay is performed entirely at room temperature.

• The sample is out of range. Use a sample that is less concentrated.
• View the Check Standards Screen to confirm that the values for the samples fall between the values of the standards (see page 18).
• Ensure that the lid is closed while reading standards and samples.
• Prepare samples and standards according to the instructions in the Qubit® assay kit you are using.
• Ensure that the assay is performed entirely at room temperature.

• The sample is out of range. 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).
•  View the Check Standards Screen to confirm that the values for the samples fall between the values of the standards (see page 18).
•  Ensure that you have prepared the Qubit® working solution correctly (1:200 dilution using the buffer provided in the kit).
• Ensure that you have prepared the standard tubes correctly (10 μL of each standard in 190 μL of Qubit® working solution).
• Ensure that the standard and sample tubes are filled to 200 μL.
• Protect the Qubit® reagent and working solutions from light.
• Select the correct Qubit® 2.0 Fluorometer assay for the Qubit® assay you are performing and calibrate the fluorometer correctly. Standards must be used in the correct order.
• Ensure that the assay is performed entirely at room temperature.

Negative fluorescence is a physical impossibility. It is an artifact from software autocorrecting for background signal. This means your reader is picking up and subtracting out background light at the cost of your data. Make sure to do a buffer-only control and assess the type of signal. You may need to switch to a different plate.