Sensitive and specific nucleic acid and protein quantification assays

Invitrogen Qubit assays use target-selective dyes that emit fluorescence only when bound to their DNA, RNA, or protein target. They are more accurate than traditional UV absorbance, which can overestimate sample concentrations due to contaminants such as salts, solvents, detergents, proteins, and free nucleotides. Fluorescence measurements are also much more sensitive than UV absorbance, and Qubit Fluorometers can accurately measure dilute samples with significantly less noise.

Qubit-Flex-and-Assays

Tip: For high-throughput quantification, consider using a microplate reader with fluorescence capabilities.. The Thermo Scientific Fluoroskan Microplate Fluorometer and Varioskan LUX Multimode Microplate Reader are compatible with Quant-iT assays to quantify the same analytes as Qubit Fluorometers and assays. For a comparison, see our RNA/DNA Quantification page.


Features

While Qubit assays differ in the analytes they measure, they share characteristics that make them selective, sensitive, and easy to use with the Qubit 4 or Flex Fluorometer.

Dyes bind selectively to DNA, RNA, or protein

More sensitive than UV absorbance and traditional Bradford protein assays

Dyes emit fluorescence only when bound to target

Minimizes effects of contaminants, including degraded DNA or RNA

Easy to perform

Simple mix-and-read format

Short incubation times

Only 2 minutes for DNA and RNA assays or 10–15 minutes for protein assays

Designed for Qubit Fluorometers

Optimized for use with the Qubit 4 and Qubit Flex Fluorometers

Comparison of assay quantification ranges

Qubit assays are available for double- and single-stranded DNA (dsDNA and ssDNA), RNA, microRNA, and protein. Qubit DNA and RNA assays are available with different quantification ranges. The HS (high sensitivity) assays quantitate the lowest levels of analyte, the BR (broad range) assays do so across a wider range, and the RNA XR (extended range) assay is ideal for abundant samples. These various detection ranges allow accurate measurement across a wide range of concentrations. As the graph shows, both Qubit HS and BR assays detect lower levels of analyte than UV absorbance.

Sample concentration ranges for Qubit assays and UV absorbance

Fluorescence-based quantification using the Qubit system is very sensitive, quantitating samples of low abundance, and measures only the intended target, even if the sample contains both DNA and RNA. Standard UV absorbance readings are not sensitive enough to detect low levels of analyte and do not distinguish between RNA vs DNA, quantitating all nucleic acid present in a sample.

Comparison of assay quantification ranges

In addition, fluorescence-based quantification using the Qubit system can distinguish between RNA and DNA in the same sample whereas UV absorbance readings typically cannot. With the total solution offered by Qubit platforms, you can be confident in your results because assays, consumables and instruments have been co-optimized for the best performance.


DNA quantification assays

Qubit dsDNA assays are available in two quantification ranges (HS and BR) and two formats (standard and 1X), both important considerations for downstream applications and processing. An assay that quantifies ssDNA and oligonucleotides is also available.

DNA quantification assays

Product

Initial sample concentration

Quantitation range

No. of assays

Cat. No.

High Sensitivity dsDNA Assays

Qubit 1X dsDNA HS Assay Kit

0.005–120 ng/µL

0.1–120 ng

100

Q33230

500

Q33231

Qubit dsDNA HS Assay Kit

0.005–120 ng/µL

0.1–120 ng

100

Q32851

500

Q32854

Qubit 1X dsDNA HS Assay Lambda Standard

500

Q33233

Broad Range dsDNA Assays

Qubit 1X dsDNA BR Assay Kit

0.2–4,000 ng/µL

4–4,000 ng

100

Q33265

500

Q33266

Qubit dsDNA BR Assay Kit

0.2–2,000 ng/µL

4–2,000 ng

100

Q32850

500

Q32853

Qubit 1X dsDNA BR Assay Lambda Standard

500

Q33263

ssDNA Assay

Qubit ssDNA Assay Kit

0.01–240 ng/µL

0.2–240 ng

100

Q10212

dsDNA quantification assays

Our Qubit dsDNA high-sensitivity (HS) and broad-range (BR) assays are highly selective for dsDNA over RNA and ssDNA. The high-sensitivity assay is designed for samples with low concentrations of dsDNA, which helps to conserve precious samples. Common contaminants such as salts, free nucleotides, RNA, solvents, detergents, or protein are well tolerated in the dsDNA HS and BR assays.

Qubit-4-results-screen
Results-plotted-on-standard-curve

Qubit dsDNA HS assay results

 

On either the Qubit 4 (shown) or Qubit Flex Fluorometer, results can be displayed either numerically or graphically. These images show our high-sensitivity (HS) dsDNA assay designed for samples with low DNA concentrations.

In this test, accuracy and precision were assessed on the Qubit 4 and Flex Fluorometers, as well as a competitor’s instrument, using the Qubit dsDNA HS and BR assays. Percent deviation and CV were both lower for the Qubit 4 and Flex instruments than the competitor’s, demonstrating higher accuracy and precision.

Accuracy and precision of Qubit Fluorometers.

Accuracy was assessed as average deviation from true concentration using the Qubit dsDNA HS Assay, while precision was evaluated as CV across replicates of various concentrations using the Qubit dsDNA BR Assay. The low deviation percentages demonstrate the accuracy of the Qubit Flex and Qubit 4 Fluorometers, while the low CV percentages demonstrate their precision, especially the Qubit Flex model. Of the three instruments tested, the competitor’s instrument had the lowest accuracy and precision.

Qubit fluorometers v Competitor Accuracy and Precision

Save time and help the environment with the Qubit 1X Assay format

Qubit 1X dsDNA assays are provided in a convenient pre-mixed format that eliminates the step of preparing the Qubit working solution. Simply add your sample or standard to the premixed solution, incubate, and read your results. This format offers a simplified, faster workflow while reducing the number of tubes in the kit (and therefore the amount of plastic used).

 

The Qubit 1X dsDNA HS assay provides the same dynamic range and limit of detection as the standard dsDNA HS assay, while the Qubit 1X dsDNA BR assay has a higher dynamic range than the standard dsDNA BR assay, achieving 4,000 ng/µL in the extended range.

 

In this test of the Qubit 1X dsDNA HS (left) and BR (right) assays over their core ranges, the measured amount of dsDNA deviated from the expected value by less than 10%, even in the presence of equal amounts of RNA.

A) Qubit 1X dsDNA HS Assay

1X-dsDNA-HS-graph

B) Qubit 1X dsDNA BR Assay

1X-dsDNA-BR-graph

Selectivity of Qubit 1X dsDNA HS (A) and BR (B) assays

Plots show the quantification results for samples seeded with known quantities of DNA and RNA vs expected values. Circles represent samples with 10 μL of DNA plus 190 μL of working solution, at varying concentrations. Squares represent samples with 10 μL of RNA and 10 μL of DNA plus 180 μL of working solution, at varying concentrations. The closeness of the circles and squares—in some instances almost indistinguishable—demonstrate that these two dsDNA assays are minimally affected by the presence of RNA.

ssDNA quantification assay

The Qubit ssDNA Assay Kit is designed to quantify long ssDNA or oligonucleotides. Purify ssDNA before performing the assay, as it can also detect dsDNA and RNA.  However, assay results will not be affected by common contaminants such as salts, short oligonucleotides (≤6 bases), free nucleotides, solvents, detergents, or protein.


RNA quantification assays

There are three Qubit RNA assays to quantify different concentration ranges of large RNA, and one assay to quantify small RNA molecules (microRNA). For an additional assay that measures the integrity and quality of your RNA sample, see the next section on the RNA IQ assay.

Large RNA quantification assays

Product

Initial sample concentration

Quantitation range

No. of assays

Cat. No.

General RNA assays

Qubit RNA HS Assay Kit

0.2–200 ng/µL

4–200 ng

100

Q32852

500

Q32855

Qubit RNA BR Assay Kit

0.5–1,200 ng/µL

10–1,200 ng

100

Q10210

500

Q10211

Qubit RNA XR Assay Kit

5–20,000 ng/µL

100–20,000 ng

100

Q33223

500

Q33224

microRNA assay

Qubit microRNA Assay Kit

0.025–150 ng/µL

0.5–150 ng

100

Q32880

500

Q32881

RNA quantification assays

To quantify RNA, you can choose among three Qubit RNA Assay kits with overlapping ranges of 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 for an accurate measurement, even if DNA is present in the sample. Common contaminants such as salts, free nucleotides, solvents, detergents, or proteins are well tolerated in these assays.

 

In this sensitivity test, both the Qubit RNA and microRNA assays (red and blue bars) measured very close to the actual concentrations of their target RNAs (red and blue lines).

Qubit RNA and microRNA assay sensitivity

Ribosomal RNA (rRNA) at the concentrations listed on the x-axis was added to samples containing 2 μg/mL siRNA. The mixtures were then assayed using the Qubit microRNA assay, the Qubit RNA assay, and the NanoDrop A260 assay, which quantifies using UV absorbance. Results from eight replicates were averaged, with standard deviations shown. The NanoDrop instrument’s (purple bars) detection limit for total RNA concentration is 1.5 μg/mL, affecting its accuracy and precision at low concentration levels. The Qubit RNA assay (red bars) accurately quantified the rRNA concentration over a broad scale. The Qubit microRNA assay (blue bars) accurately quantified the 2 μg/mL siRNA concentration, while accuracy was mildly affected as rRNA increased from 2 to 5 times that amount. The blue and red trendlines indicate the actual concentrations of siRNA and rRNA in the samples, respectively.

Qubit RNA and microRNA assays

microRNA quantification 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 messenger RNA (mRNA) (>1,000 bp).

 

Small RNA in pure samples has reproducibly been quantified at levels as low as 0.5 ng in the assay tube. The assay detects all types of small RNA, including microRNA and short interfering RNA (siRNA), both single- and double-stranded. Because it does not detect nucleotides, it can distinguish small mRNA particles from completely degraded RNA.


RNA integrity and quality (IQ) assay

The Qubit RNA IQ Assay was developed to quickly assess the integrity and quality (IQ) of an RNA sample. It uses two dyes: one that binds to large, intact, and/or structured RNA, the other to small, degraded RNA. No special handling, tedious sample preparation, or waiting for results is required.

RNA quality assay

Product

No. of assays

Cat. No.

Qubit RNA IQ Assay Kit

75

Q33221

275

Q33222

Qubit RNA IQ Standards

1 set

Q33235


Protein quantification assays

The Qubit Protein Assay Kits are designed to make protein quantification easy and accurate. Addressing overlapping concentration ranges, the two kits together are accurate for initial sample concentrations from 12.5 μg/mL to 20 mg/mL and exhibit low protein-to-protein variation. Qubit protein assays are easy to perform and require only 10–15 minutes of room temperature incubation, eliminating long incubation periods or exposure to elevated temperatures.

Protein quantification assays

Product

Initial sample concentration

Quantitation range

No. of assays

Cat. No.

Qubit Protein Assay Kit

12.5 µg/µL–5 mg/µL

0.25–5 µg

100

Q33211

500

Q33212

Qubit Protein BR Assay (Qubit 4 only)

100 µg/µL–20 mg/µL

1–400 µg

100

A50668

500

A50669

 


System verification assays

Invitrogen Qubit System Verification Kits are fast, easy-to-use, reagent-based assays that test the performance of the Qubit 4 and Qubit Flex Fluorometers. The three components include 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).

System-Verification

System verification assays

Product

Initial sample concentration

Quantitation range

No. of assays

Cat. No.

Qubit 4 System Verification Assay Kit

N/A

N/A

100

Q33237

Qubit Flex System Verification Assay Kit

N/A

N/A

100

Q33254

 

Note: Each Qubit system verification assay can be used only with its specified Qubit Fluorometer and not with the other.


MyQubit custom and preconfigured assays

Custom MyQubit assays allow you to create additional assays for your Qubit Fluorometer in minutes. Any reagent or assay that is spectrally compatible with the Qubit hardware can be adapted for use with the Invitrogen Qubit Fluorometer. Several preconfigured MyQubit assays are also available for download.

Custom-Assay

Create new MyQubit assays

MyQubit assays bring customized fluorescence assays right to your benchtop, providing a reliable platform for many quantitation needs, from laboratory research and quality control to process monitoring and beyond. You can add new assays to your Qubit 3.0 and 4 Fluorometer via simple download and transfer of .qbt files using a USB drive.

MyQubit Assay Design Tool

The MyQubit Assay Design Tool helps you create custom MyQubit assay .qbt files that you can upload to your Qubit 3.0 and 4 Fluorometer.

My Qubit Assay Design Tool

Download preconfigured MyQubit assays

As examples of the kinds of custom MyQubit assays you can create, we have developed new MyQubit assays for several commonly quantitated analytes using Amplex Red reagents. When you transfer the assay .qbt files to your Qubit Fluorometer via a USB drive, they will be available alongside the factory-loaded Qubit assays, extending the capabilities of your Qubit Fluorometer.

Preconfigured MyQubit assays available for download

Assay

Download for Qubit 3.0 and 4

Download for Qubit 2.0*

PDF manual

Amplex Red Cholesterol Assay

.qbt file

.qbt file

Amplex Cholesterol Assay manual

Amplex Red Galactose Assay

.qbt file

.qbt file

Amplex Red Galactose Assay manual

Amplex Glucose Assay

.qbt file

.qbt file

Amplex Glucose Assay manual

Amplex Red Glutamic Acid Assay

.qbt file

.qbt file

Amplex Red Glutamic Acid Assay manual

Amplex Red Peroxide Assay

.qbt file

.qbt file

Amplex Red Peroxide Assay manual

Amplex Red Sucrose Assay

.qbt file

.qbt file

Amplex Red Sucrose Assay manual

 

* The Qubit 2.0 Fluorometer requires a USB flash drive of 2 GB or less, such as the one provided with the instrument, to transfer MyQubit files.

 

To download the .qbt file, click on the .qbt file link of interest in the table.  It will automatically download to your computer. Make sure you click the correct link for your Qubit model. Consult the assay’s PDF manual for instructions on uploading the .qbt file to your Qubit Fluorometer.


Ion Sphere Quality Control Kit

In the Ion Torrent method of next-generation sequencing (NGS), multiple copies of each amplicon are created on an Ion Sphere Particle (ISP) during template preparation. Before sequencing DNA or RNA, it’s important to know the amount of template positive ISPs.

 

The Ion Sphere Quality Control Kit enables quick and easy assessment of enriched ISPs on your Qubit  Fluorometer prior to performing an NGS run on the Ion Personal Genome Machine (PGM) or Ion GeneStudio SystemAll 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.

Ion Sphere assay

Product

No. of assays

Cat. No.

Ion Sphere Quality Control Kit (Qubit 4 only)

20

4468656

 

The Qubit 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 a Qubit fluorometer helps to determine the likelihood of a successful sequencing run, ultimately saving time and expense during subsequent sequencing.

Ion Sphere Particle assay principle

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, while the A sequence is present only in the template. Using Invitrogen Alexa Fluor dyes, the ratio of fluorescence signals from nucleotides labeled with Alexa Fluor 647 (red) and Alexa Fluor 488 (green) dyes is used to determine the relative quantity of template-positive particles.

Ion Sphere Quality Control Kit Principle

For Research Use Only. Not for use in diagnostic procedures.