Sensitive and specific nucleic acid and protein quantification assays
Invitrogen Qubit assays utilize target-selective dyes that emit fluorescence when bound to DNA, RNA or protein, unlike traditional 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 and traditional Bradford Assays
- Dyes emit fluorescence only when bound to the target, minimizing the effects of contaminants—including degraded DNA or RNA
- All assays are easy to perform using a simple mix-and-read format
- Short incubation times of only 2 minutes required for DNA and RNA assays or 10-15 minutes for protein assays
- 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 typically 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.
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 (ng/uL)||Quantitation range (ng)||Cat. No.||No. of assays|
|High Sensitivity Assays|
|Qubit 1X dsDNA HS Assay Kit*||0.005 – 120||0.1 to 120||Q33230||100|
|Qubit dsDNA HS Assay Kit||0.005 – 120||0.1 to 120||Q32851||100|
|Qubit 1X dsDNA HS Assay Lambda Standard||—||—||Q33233||500|
|Broad Range Assays|
|Qubit 1X dsDNA BR Assay Kit*||0.2 to 4000||4 to 4000||Q33265||100|
|Qubit dsDNA BR Assay Kit||0.2 to 2000||4 to 2000||Q32850||100|
|Qubit 1X dsDNA BR Assay Lambda Standards||—||—||Q33263||500|
* Ready-to-use formulation of the dsDNA HS and BR assay kits that does not require mixing of buffer and reagent is stable for up to 6 months.
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
To quantify RNA, there are three Invitrogen Qubit RNA Assay kits which include:
- Qubit RNA HS Assay Kit - High Sensitivity
- Qubit RNA BR Assay Kit - Broad Range
- Qubit RNA XR Assay Kit - Extended Range
The assays are accurate for initial sample concentrations from as little as 250 pg/μL to 10,000 ng/μL.
These kits are highly selective for RNA over double-stranded DNA (dsDNA). Unlike other RNA assays, they do not require DNase if DNA is present in the sample for an accurate measurement. Additionally, common contaminants such as salts, free nucleotides, solvents, detergents, or proteins are well tolerated in the assays.
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 6). 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 or Qubit Flex 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 7).
Figure 6. 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 (Cat. No. Q33238). Relative fluorescent units (RFUs) (A) and IQ#’s were plotted (B) for these samples.
Figure 7. 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. Two assay kits are available:
- Qubit Protein Assay
- Qubit Protein BR Assay
Together these assays are accurate for initial sample concentrations from 12.5 μg/mL to 20 mg/mL and exhibit low protein-to-protein variation. Common contaminants, such as reducing reagents (DTT, β-mercaptoethanol), salts, free nucleotides, amino acids, solvents, DNA, and detergents (Qubit Protein BR assay only) are well tolerated in the assays; 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 8. 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 or protein samples?
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.
Save time with the Qubit 1X Assay Format
We offer the Qubit 1X Assays that eliminate the step of preparing the Qubit working solution. The Qubit 1X dsDNA HS assay provides the same dynamic range and limit of detection as the standard assay, while the Qubit 1X dsDNA BR assay has a higher dynamic range than the standard assay achieving 4,000 ng/µL in the extended range.
This format offers a simplified workflow while reducing the 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.
Technical and application notes
Posters and presentations
For Research Use Only. Not for use in diagnostic procedures.