Quantification of Nucleic Acids Using Qubit Fluorometer
How does this technology quantify nucleic acids?
The Qubit Fluorometer utilizes specially formulated dyes that bind specifically to DNA or
RNA.
The Nanodrop utilizes UV absorbance and compares the ratio of absorbance between
260nm and 280nm (260/280).
Which technology will provide me with both RNA and DNA concentrations?
The Qubit Fluorometer has the capability to provide DNA OR RNA concentrations from the
same sample.
The Nanodrop can only provide nondiscriminatory concentrations for both DNA and RNA;
in other words, both DNA and RNA concentrations are combined in resulting concentration
readings (see NanoDrop Info PDF )
What are the minimal concentrations possible with the Qubit Fluorometer and
NanoDrop Spectrophotometer?
Qubit Fluorometer has an accurate quantitative range of 10pg/ul to 1ug/ul. This
technology has an input volume of 1-20ul. *If your sample has a very low concentration
additional sample input to the assay allows for quantification.
NanoDrop Spectrophotometer has the potential to significantly overestimate concentration
measurements. Variation can be high with the same sample tested multiple times. Samples
may range from 2ng/ul to 3.7 ug/ul. Sample input volume ranges from 1-2ul (See
NanoDrop Info PDF).
Sample requirements for Qubit quantification?
Only drop off an aliquot(s) of your sample! Not the Stock!
Ensure all samples, to the best of your knowledge, fit within a specific assay’s range.
How do contaminants affect NanoDrop concentrations?
Our facility has observed the effects of contaminants on the concentration reading
provided by a NanoDrop spectrophotometer. As an example, TRIzol has been shown to
“shoulder” concentration readings over 200%. In the below image we can see the effects of
TRIzol (TRZ) on an RNA sample. An RNA sample at 48ng/ul is projected to be 183.9ng/ul
after of TRIzol contamination. A peak at 272nm is amplifying the signal of the 260nm peak.
RNA
TRZ
What are the limits of contamination?
Downstream applications sometimes require samples to be clear of any buffers, salts,
phenols, or proteins to continue. The use of the NanoDrop may sometimes lead to
erroneous quantification caused by high concentrations of contaminants. Like the
NanoDrop, the Qubit Fluorometer may be affected by these contaminants. However the
Qubit Fluorometer is more forgiving as the system uses dyes to “label” DNA or RNA in
order to determine the concentration. While it is beneficial to have a system that displays
high sample specificity, it is important to note that high concentrations of contaminants
will effect quantification. Contaminant restraints differ between the types of assay. The
following tables may aid in determining whether your sample can be read accurately on the
fluorometer.
How do I submit a sample for Qubit quantification?
1.
2.
3.
4.
Go to https://my.ilabsolutions.com/service_center/3129/?tab=services
Click the “VGN Microarray Facility” link under Sample Submission and Laboratory Services.
Select the “Initiate Request” for Qubit Nucleic Acid Quantification.
Select
a. The Person or the investigator who the service is for
b. Click Proceed
c. Select the Lab
Your Name Here
5.
Enter the number of samples you want quantified.
6.
Pay close attention to your Assay Kit Selection options and pick the one that fits your needs.
7.
Now select the number of samples and click the
8.
Enter your sample information
a. Sample ID: What is written on the tube?
b. Input Volume: 1-10ul *enter 1 for most samples*
c. Max. Volume: Maximum volume available for quantification
i. Sometimes 1ul is not enough for accurate quantification. Additional sample is sometimes
needed. What is the maximum volume you can provide for quantification
d. Isolation Method: used to help troubleshoot if necessary
9.
Now select your chartstring
10. Now that you’ve filled out the form, simply click
you’re done!
and
and
How Do I print a copy of my service request?
After you request your quantification service, locate the pencil icon
hand of the screen, click print, then select WEB.
on the right
Where do I put my samples?
Ensure that the names on the tubes match your order form “Sample ID”!
Ensure ONLY ALIQUOTS are submitted, NO STOCKS!
Place your samples in the Labeled refrigerators within the entry area of the AGTC Core
Facility
Qubit RNA Quantification for RNA samples (-20°C)
Qubit DNA Quantification for DNA samples (4°C)