Sensitive and specific nucleic acid quantification assays
Invitrogen Qubit assays utilize target-selective dyes that emit fluorescence when bound to DNA, RNA or protein. Unlike UV absorbance, which can overestimate sample concentrations due to contaminants in the sample such as salts, solvents, detergents, proteins, free nucleotides.
Fluorescence measurements are also much more sensitive than UV absorbance, and Invitrogen Qubit Fluorometers are able to accurately measure dilute samples with significantly less noise.
Features of Qubit assays
- Dyes bind selectively to DNA, RNA, or protein making the assay more sensitive than UV absorbance (Figure 1)
- Dyes emit fluorescence only, minimizing the effects of contaminants—including degraded DNA or RNA—on the result
- All assays performed using the same general protocol with a simple mix-and-read format
- Incubation times of only 2 minutes required for DNA and RNA assays or 15 minutes for protein assay
- Optimal use with the Invitrogen Qubit 4 Fluorometer and the Invitrogen Qubit Flex Fluorometer
Quantification range comparison between the Qubit assays and UV absorbance
We’ve developed Qubit assays for dsDNA, ssDNA, RNA, miRNA, and protein. Figure 1 shows the effective quantifiable range for each Qubit nucleic acid assay as compared to the effective range for UV absorbance readings. It's important to note that fluorescence-based quantification using the Qubit system distinguishes between RNA and DNA in the same sample whereas UV absorbance readings do not. With the total solution offered by Qubit platforms, you can be confident in your results because assays, consumables and instruments have been optimized together for best performance.
Figure 1. Comparison of sample concentration ranges for Qubit assays and UV absorbance measurements. UV absorbance readings are not selective for RNA vs DNA. The Qubit 4 Fluorometer and all Qubit assays are optimized for use together.
dsDNA quantification assays
Our Qubit dsDNA high-sensitivity (HS) and broad-range (BR) assays are highly selective for double-stranded DNA (dsDNA) over RNA and single-stranded DNA (ssDNA). In addition to our broad range assay, we offer a high-sensitivity assay for samples with a low concentration of dsDNA, making it ideal for precious samples (Figures 2 and 3). Common contaminants such as salts, free nucleotides, RNA, solvents, detergents, or protein are well tolerated in the dsDNA HS and BR assays.
Figure 3. Accuracy and precision of the Qubit Quantification Platform. Ten replicates of lambda DNA at concentrations from 0.01 to 10 ng/μL were assayed using the Qubit dsDNA HS Assay on the Qubit Fluorometer according to the standard kit protocol. The same concentrations of DNA were measured in 10 replicates using a microvolume spectrophotometer, and results were compared for both accuracy and precision. Each bar represents the average of 10 replicates. Error bars represent the standard deviations of the 10 replicates. The concentrations indicated are the concentrations of DNA in the starting samples, before dilution in the Qubit assay tubes.
|Product||Initial sample concentration||Quantitation range||Cat. No.||No. of assays|
|Broad Range Assays|
|Qubit dsDNA BR Assay Kit||100 pg/µl–1,000 ng/µl||2–1,000 ng||Q32850||100|
|High Sensitivity Assays|
|Qubit 1x dsDNA HS Assay Kit*||10 pg/µl to 100 ng/µl||0.2–100 ng||Q33230||100|
|Qubit dsDNA HS Assay Kit||10 pg/µl to 100 ng/µl||0.2–100 ng||Q32851||100|
* Ready-to-use formulation of the dsDNA HS assay kit that does not require mixing of buffer and reagent is stable for up to 1 year.
Save time with the Qubit 1X dsDNA HS Assay
We now offer the Qubit 1X dsDNA assay which provides the same dynamic range and limit of detection as the Qubit dsDNA HS assay while eliminating the step of preparing the Qubit working solution by diluting the reagent with the buffer. This offers a simplified workflow while reducing the number of tubes in the kit, therefore reducing the amount of plastic used.
Simply add your sample or standard to the premixed solution, incubate, and read your results.
ssDNA and oligos quantification assays
The Qubit ssDNA Kit is ideal for quantitating single-stranded DNA or oligonucleotides. Common contaminants such as salts, free nucleotides, solvents, detergents, or protein are well tolerated in the assay.
RNA quantification assays
The Invitrogen Qubit RNA BR (broad range) and HS (high sensitivity) Assay Kits are highly selective for RNA over double-stranded DNA (dsDNA) and unlike other RNA assays do not require DNase if DNA is present in the sample for an accurate measurement.
The assays are accurate for initial sample concentrations from as little as 250 pg/μL. Common contaminants, such as salts, free nucleotides, solvents, detergents, or protein are well tolerated in the assay.
The Invitrogen Qubit microRNA Assay Kit is designed for easy and accurate quantification of small RNA (17–25 nt or bp) such as microRNA, even in the presence of common contaminants such as salts, free nucleotides, solvents, detergents, and protein. The assay is highly selective for small RNA over rRNA or large mRNA (>1,000 bp).
We have been able to reproducibly quantify small RNA in pure samples at levels as low as 0.5 ng in the assay tube. The assay detects all types of small RNA, including microRNA and siRNA, both single stranded and double stranded.
Read the BioProbes 70 article—Fluorescence-Based Small RNA Quantification for Both Conventional and High-Throughput Assays
The Qubit RNA IQ Assay utilizes two unique dyes—one that binds to large, intact and/or structured RNA, the other selectively binds to small, degraded RNA (Figure 5). Together they are able to quickly assess the quality and integrity of an RNA sample. To use, simply add your samples to the RNA IQ working solution, then measure on the Qubit 4 Fluorometer. Results are presented as a total value for the RNA sample integrity and quality or RNA IQ# and as the calculated % of large and small RNA in the sample (Figure 6).
Figure 5. Selectivity of the RNA IQ reagents for large and small RNA. Triplicate samples containing 100 ng/mL rRNA (E. coli) and varying amounts of siRNA (0 to 50 ng/μL) were assayed with the Qubit RNA IQ assay (Q33221, Q33222) on the Qubit 4 Fluorometer. Relative fluorescent units (RFUs) (A) and IQ#’s were plotted (B) for these samples.
Figure 6. A proprietary algorithm is used to report a quality score representative of the ratio of small and large and/or structured RNA in the sample. The score is a value from 1 to 10, similar to other RNA quality scores. With the Qubit RNA IQ Assay, a small number indicates that the sample consists of mainly small RNA.
The Invitrogen Qubit Protein Assay Kits are designed to make protein quantification easy and accurate. The assay is accurate for initial sample concentrations from 12.5 μg/mL to 5 mg/mL and exhibits low protein-to-protein variation.
Common contaminants, such as reducing reagents (DTT, β-mercaptoethanol), salts, free nucleotides, amino acids, solvents, and DNA (but not detergents) are well tolerated in the assay; some very slight modifications for the procedure are required for other contaminants.
The Ion Sphere assay on the Qubit 4 Fluorometer enables quick and easy assessment of Ion Sphere Particles (ISPs) prior to performing a sequencing run on the Ion Personal Genome Machine (PGM) Sequencer using the Ion Sphere Quality Control Kit. All ISPs bind a complimentary, green-fluorescent Invitrogen Alexa Fluor 488 dye–labeled oligonucleotide, whereas only those beads that are template-positive bind a complementary red-fluorescent Invitrogen Alexa Fluor 647 dye–labeled oligonucleotide. The Qubit 4 Fluorometer measures the fluorescence emission of each dye conjugate, and an Excel™ file template is used to calculate the percentage of template-positive ISPs from the ratio of the Alexa Fluor 647 dye signal to the Alexa Fluor 488 dye signal. This quality-control step using the Qubit fluorometer helps determine the likelihood of a successful sequencing run, ultimately saving time and expense during subsequent sequencing.
Figure 7. The Ion Sphere Particle (ISP) assay principle using Invitrogen Alexa Fluor dyes. The B nucleic acid sequence is present in both the ISP primer and the template and is used to drive the amplification of the template onto the bead; the A sequence is only present in the template. The ratio of fluorescence signals from Alexa Fluor 647– and Alexa Fluor 488 dye–labeled oligonucleotides is used to determine the relative amount of template-positive particles.
Create your own assays using MyQubit
Since the Qubit fluorometer is operated by simple components, creation of additional applications is as straightforward as matching spectral compatibility with the LEDs and emission filters used. Use the MyQubit assay design tool to create your own assays to run on the Qubit fluorometer.
You can add new assays to your Qubit fluorometer via simple download and transfer of .qbt files using a USB cable or USB drive.
Go to MyQubit to download preconfigured assays or create new ones.
Invitrogen Qubit System Verification Kits are fast, easy-to-use, reagent-based assay that tests the performance of the Qubit 4 Fluorometer. The kit consists of three components: a blank reagent solution, a green fluorescent reagent, and a far-red fluorescent reagent. The green fluorescent reagent is designed to test the blue excitation (470 nm LED; 430-495 nm excitation filter) and green fluorescence detection (510-580 nm emission filter) on the fluorometer, while the far-red fluorescent reagent is designed to test the red excitation (635 nm LED; 600-645 nm excitation filter) and far-red fluorescence detection (665-720 nm emission filter).
Note: The Qubit 4 System Verification Assay Kit can only be used with the Qubit 4 Fluorometer. The Qubit Flex System Verification Assay Kit can only be used with the Qubit Flex Fluorometer.
It is important to use tubes without autofluorescence, which introduces background signal and error. For best results, use the tubes that were optimized with the Qubit fluorometers.
Need higher throughput for your nucleic acid samples?
Qubit assays are ideal when the number of samples you measure at a time is low enough not to warrant a microplate reader.
For larger sample batches requiring a fluorescence microplate reader, use Invitrogen Quant-iT assay kits and reagents for use with microplate readers for nucleic acids or protein quantification. Quant-iT assays are also compatible with the Qubit Fluorometer.
Technical and application notes
Posters and presentations
For Research Use Only. Not for use in diagnostic procedures.