Fast, accurate, specific quantification of DNA, RNA, and protein

Quantification of nucleic acids and proteins is important for downstream applications like next generation sequencing (NGS), PCR, transfection, western blotting, immunoassays, and more. Qubit 4 and Qubit Flex Fluorometers quantify these targets by detecting fluorescent dyes in a variety of Qubit Assays that bind very specifically to their target molecules. The measurements are accurate, precise, and sensitive and the process is quick and easy.

Qubit fluorescence quantification technology

Qubit Fluorometers detect fluorescent dyes in Qubit Assays that are highly specific to a target molecule of interest in your samples. These dyes emit fluorescence only when bound to their targets, even at low concentrations, so the readings are also highly sensitive. The dyes are absorbed within minutes and can be read in seconds by Qubit Fluorometers, which interpolate the readings onto a standard curve.

Fluorescent dyes selectively bind to DNA, RNA, or protein

Qubit fluorometers use specialized curve-fitting algorithms to develop a calibration curve using standard samples with a known concentration. An unknown sample concentration of DNA, RNA, or protein is calculated by comparing the relative fluorescence units (RFUs) of the sample to the RFUs of the standards. The detection limits of the measurements are specific to each assay.

Qubit fluorescence technology is so sensitive that Qubit Assays require as little as 1 µL of sample, even if the sample is very dilute. This makes them ideal for quantifying precious samples.

Accurate, specific, and sensitive

Both the Qubit 4 and Qubit Flex Fluorometers give fast, accurate quantification measures of the specific molecule they are designed to detect, even in extremely small amounts. For example, Qubit provides DNA assays to detect either double- or single-stranded DNA molecules (dsDNA or ssDNA). These assays are highly selective for their target DNA type, even in the presence of RNA or the other DNA type, or of common contaminants such as salts, free nucleotides, solvents, detergents, and protein.

DNA quantification

In this test, the Qubit dsDNA HS (High Sensitivity) Assay demonstrated a linear detection range of 0.2–100 ng and selectivity for double-stranded DNA (dsDNA), even in the presence of an equal mass of RNA.

Qubit dsDNA HS Assay sensitivity and selectivity

The Qubit dsDNA HS Assay reported accurate, linear results for known, ascending quantities of dsDNA (green circles), even in low amounts (inset, enlarging the very low end of the scale). These DNA-specific measurements were only slightly affected by RNA added alone (red triangles) or in combination with DNA (blue squares).

Accuracy and precision were assessed on the Qubit 4 and Flex Fluorometers, as well as a competitor’s instrument, using the Qubit dsDNA HS Assay and the Qubit dsDNA BR (Broad Range) Assay, which is optimized to detect higher dsDNA concentrations. Both accuracy and precision were higher for the Qubit instruments than the competitor’s, with the Qubit Flex instrument offering increased throughput.

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 coefficient of variation (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.

In an independent 2013 PLoS One publication, Simbolo et al. reported that Qubit fluorometric quantification was a reliable and cost-effective method to qualify various DNA preparations for NGS, including those derived from frozen tissue and FFPE samples. Of the instruments they tested, their data showed that DNA quantification results obtained using the Qubit Fluorometer were highly reproducible and were consistent with qPCR data for DNA quantity—even for partially degraded DNA from FFPE samples.¹

 

1 Simbolo M, Gottardi M, Corbo V, et al. DNA qualification workflow for next generation sequencing of histopathological samples. PLoS ONE. 2013; 8: e62692.  Full text

RNA quantification

Qubit provides RNA assays to detect both large, intact RNA molecules (such as rRNA or large mRNA) and small, intact RNA molecules (such as microRNA and siRNA). These assays are highly selective for their RNA type, even in the presence of DNA or the other RNA type, or of common contaminants. In this sensitivity test, both the Qubit RNA and microRNA assays measured quite close to the actual concentrations of their target RNAs.

Qubit RNA and microRNA assay accuracy and selectivity

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.

More sensitive and selective than UV absorbance

Qubit Fluorometers are orders of magnitude more sensitive than UV absorbance, an alternative method that can quantify nucleic acids due to their absorption of ultraviolet light at 260 nm. Historically, spectrophotometers that measure UV absorbance could not discern the differences among DNA, RNA, free nucleotides, excess salts, and other organic compounds, which all absorb at that wavelength.

 

Although advanced software algorithms on UV-Vis (ultraviolet/visible) instruments that analyze more spectrum can successfully differentiate DNA from RNA, they still cannot distinguish dsDNA from ssDNA or rRNA from microRNA. Moreover, UV spectrophotometry often does not have the sensitivity to accurately measure low concentrations of DNA and RNA.

 

Note: Although UV absorbance is not as sensitive or selective as fluorometry at quantifying nucleic acids or proteins, it is excellent at detecting impurities in a sample. Many labs use both of these technologies for different purposes. Compare the two and check out our NanoDrop spectrophotometers at our RNA/DNA Quantification page.

Qubit dsDNA accuracy and sensitivity vs UV absorbance.

Ten replicates of lambda DNA at known concentrations from 0.01 to 10 ng/μL were assayed using the Qubit dsDNA HS Assay on the Qubit Fluorometer (blue bars) according to the standard kit protocol. The same concentrations of DNA were measured via UV absorbance in 10 replicates using a microvolume spectrophotometer (blue bars), and results were compared for both accuracy and precision. Each bar represents the average of 10 replicates, while error bars (almost indiscernible for Qubit measurements) represent their standard deviations. The x-axis scale represents the known concentrations of DNA in the starting samples, before dilution in the Qubit assay tubes, while the y-axis scale shows the measured concentrations. The Qubit Fluorometer measurements were more accurate (y » x), sensitive (low concentrations, inset), and precise (much smaller error bars) than the UV absorbance measurements.

“Our data strongly suggest that the ideal workflow to quantify DNA from histopathological samples as suitable for NGS is to first assess the presence of contaminants in the sample with NanoDrop, and subsequently use Qubit to quantify the dsDNA.”

 

DNA qualification workflow for next generation sequencing of histopathological samples²

Simbolo, Gottardi, Corbo, et al. (2013)

2. Simbolo M, Gottardi M, Corbo V, et al. DNA qualification workflow for next generation sequencing of histopathological samples. PLoS ONE. 2013; 8: e62692. Full text

Simple and fast

Qubit Fluorometers are simple and easy to operate: just follow the detailed instructions that appear on the screen. There’s even an onboard calculator to determine the exact amount of dye and buffer to use, depending on the number of samples and standards you are testing. The video demonstrates the simple and intuitive process.

 

After you’ve isolated your DNA, RNA,  or protein samples, mix them with dye and buffer in the recommended proportions and pipet them into the assay tubes or tube strips. In the same way, prepare your two (or three) standards, which you can use for multiple samples. Once you start the test, it takes only a few seconds for the instrument to report the results.

 

You can then export the results to a Thermo Fisher Connect Cloud account via WiFi, or transfer a standard CSV file to a computer via a USB drive or cable.

 

If you have more than a few samples, use the Qubit Flex Fluorometer to increase your throughput. The graph shows time to data, including sample prep and measurement, for a given number of samples with the Qubit 4 or Flex Fluorometer as well as a competitor’s instrument.

The Qubit Flex Fluorometer reduces time to data by up to 50%

A time study comparing the Qubit Flex Fluorometer to the Qubit 4 Fluorometer and another supplier’s fluorometer, using the Qubit 1X dsDNA HS Assay Kit, showed time-to-data reduced by up to 50% with up to 96 samples.

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 Qubit Assays. For a comparison, see our RNA/DNA Quantification page.

Onboard calculators for sample preparation and downstream applications

All Qubit Fluorometers feature an onboard Reagent Calculator, which helps you determine how much reagent and buffer to use in preparing your working solution, depending on the number of samples and standards you are testing. It can be used to determine master mix volumes for samples and standards.

Onboard Reagent Calculator

The Reagent Calculator will figure out the volume of dye reagent and buffer needed to prepare sufficient working solution for the number of samples and standards you are testing. You can even include overage to make sure you don’t run short.

The Qubit Flex Fluorometer offers three additional calculators to streamline your workflow. On the sample preparation side, for the assay you’ve selected, the Assay Range Calculator displays the core sample concentration range for which it is most accurate, as well as extended low and high ranges, based on your sample volume.

Assay Range Calculator

This calculator helps you choose the Qubit assay that will offer the best accuracy for the amount and concentration of sample available.

Molarity Calculator

Use this calculator to quickly determine the molarity of your sample from its concentration and approximate length in base pairs.

Normalization Calculator

This calculator quickly determines the amount of sample and buffer needed to normalize all samples to the same concentration, mass, or molarity and volume.

To aid your transition to downstream applications, particularly NGS, two calculators help you manage assay sample data and perform common unit conversions and dilutions. The Molarity Calculator allows you to determine the molarity of a sample based on nucleic acid length and the measured concentration. For sequencing applications, the Normalization Calculator replaces the spreadsheet often used to normalize samples during library preparation. For each run, it recommends how much sample and buffer to add to reach a desired, normalized mass, concentration, or molarity. The results of both calculators can easily be exported directly to a Thermo Fisher Connect Cloud account via Wi-Fi, or in a standard CSV file to a computer or other device via USB drive or Ethernet cable.

21 CFR Part 11 compliance support software

The Qubit Flex SAE Software Solution is an optional software module that supports with FDA Title 21 CFR Part 11 regulations for electronic record-keeping and electronic signature.

21 CFR part 11 compliance is composed of both procedural and technical requirements.  

  • Procedural requirements are the standard operating procedures established by the end user’s institution
  • Technical requirements are the functional characteristics of the compliance management software used

Satisfying the technical requirements does not guarantee 21 CFR part 11 compliance alone. Compliance is the consequence of both the institution’s work process and systems used.

 

Capabilities of the software include restriction of unauthorized users, password policies and expiration dates, defined user permissions and roles, audit logs and audit reports, and on-board e-signature workflows. All Qubit Flex Fluorometers (operating firmware v.1.7.0 or later) can run in SAE Mode after purchase of the Qubit Flex SAE Software License. To achieve 21 CFR Part 11 compliance, your institution will also need to establish and document standard operating procedures.

Components

The Qubit Flex SAE Software Solution comprises three components that communicate with each other.

  1. SAE Software is used to configure the Security, Audit, and Electronic-signature (SAE) settings for the Qubit Flex Fluorometer. The SAE Software consists of the SAE Administrator Console (installed on a networked computer server with a static IP address) and the SAE Application Profile.
  2. SAE License is used to activate the SAE settings for the Qubit Flex Fluorometer.
  3. SAE Mode runs on the Qubit Flex Fluorometer and connects remotely to the SAE Administrator Console.

Component 1: SAE Software–SAE Administrator Console

The SAE Administrator Console is the component of the Qubit Flex SAE Software Solution for 21 CFR Part 11 Compliance Support that allows configuration of the Qubit Flex instrument to meet user-specific requirements. It is modular and can support other Invitrogen instruments, so the Qubit Flex Application Profile must be installed on the SAE Admin console before SAE settings on the Qubit Flex instrument can be used. Once the Qubit Flex Application Profile software is installed on the SAE Administrator Console, an SAE server (network computer) is used to connect to the Qubit Flex instrument. The SAE Admin Console must operate from a network computer with a static IP address. 

Interface view of the SAE Admin Console and the Qubit Flex Application Profile.

Component 2: SAE License

The Qubit Flex SAE Software License for 21 CFR Part 11 is for use with the Qubit Flex Fluorometer and supports compliance with 21 CFR Part 11 FDA guidelines for security, auditing, and e-signatures (SAE) using the SAE Admin Console. One SAE Software License is required to activate SAE Mode on each Qubit Flex Fluorometer. For the license to be activated, the Qubit Flex must be updated to software version 1.7.0 or later.

Component 3: SAE Mode

When SAE Mode is enabled, the Qubit Flex instrument connects to the SAE Admin Console via the SAE server. Security, Audit and E-signature settings, as defined in the SAE Admin Console, are implemented on the Qubit Flex Fluorometer.

 

Features include restriction of unauthorized users to the system, password policies and expiration dates, defined user permissions and roles, through audit logs and audit reports, and on-board e-signature workflows. All Qubit Flex Fluorometers (operating firmware v.1.7.0 or later) can run in SAE Mode through purchase of the Qubit Flex SAE Software License.

Enabling SAE mode on the Qubit Flex Fluorometer

Functionality

Functionality of the SAE Administrator Console

System security  Controls user access to the software through user IDs, passwords, roles, and permissions. Three default user roles are provided, one with full privileges (Administrator), and one with some privileges removed (Scientist). Default user roles can be edited, and additional user roles and permissions can be created. 
Auditing
 Tracks actions performed by users, and changes to the SAE Admin Console settings. When auditing is enabled, the software silently audits actions that result in changes to data or to the instrument. The option to require users to enter an audit reason (a reason for performing an action or making a change) is possible.  The auditing function provides reports for audited SAE Admin Console changes and actions. 
Electronic signature (e-signature)  Determines the functions for which users are required to provide a username and password. You can configure the e-signature event to require multiple signatures and to require users with specific permissions to sign. 

System security

Access is restricted to authorized personnel via user ID and password. 

Interface of the SAE Administrator Console showing new user account creation.

Auditing

The auditing function of the SAE Admin Console tracks actions by users and any changes to SAE settings. Actions tracked silently include signing in and out, data generation, and data exports.

SAE Admin Console audit history.

The Audit History tab of the SAE Administrator Console (1) lists action, system configuration, and application object records. For each event, it shows the (2) date and user, (3) application, (4) action, and (5) comments including object ID. You can also (6) export the records in readable or printable (.txt or PDF) format.

Within the auditing function, there are options to select whether specific user actions will be audited as silent, optional, or required. In addition, reports can be generated for user actions, SAE module changes, and software or instrument actions and events.

Electronic signatures (e-signatures)

The e-signature function of the SAE Admin Console determines the functions for which users must fulfill signature requirements.  

SAE Admin Console e-signature settings.

The e-Signature tab of the SAE Administrator Console lets you (1) customize the meanings of e-signatures, (2) choose how many signatures are required by meaning and role, and (3) choose the type of data signed for specific meanings.

Within the e-signature function, you can configure how many users in different roles must sign each e-signature event. You can also create separate meanings and customize the signature requirements for each.

Ordering information

To use the Qubit Flex SAE Software Solution with your Qubit Flex Fluorometer, you can purchase a software license independently or as part of a bundled Qubit Flex instrument and software license package.

If you already have a Qubit Flex Fluorometer, check that your instrument has firmware version 1.7.0 or later to enable this option.

Instructions for generating a license key is in the SAE User Guide.

Broad range of assays and applications

Quantification of nucleic acids is important for downstream DNA and RNA applications like NGS, RNA sequencing, PCR, qPCR, cloning, plasmid preparation, and transfection. Quantification of proteins is important for applications like protein electrophoresis, western blotting, mass spectrometry, and immunoassays.

 

Invitrogen Qubit Assays use target-selective dyes that emit fluorescence when bound to DNA, RNA, or protein. Fluorescence measurement is both more sensitive and more specific than other methods like UV absorbance or Bradford assays, which can overestimate sample concentrations due to contaminants such as salts, solvents, detergents, proteins, and free nucleotides.

 

There are Qubit assays for a broad range of quantification applications:

DNA quantification

Qubit DNA assays are highly specific for either dsDNA across two different concentration ranges, or ssDNA and oligonucleotides.

RNA quantification

Qubit RNA assays measure large rRNA and mRNA molecules across three different concentration ranges, or small RNA molecules such as microRNA.

RNA Integrity and Quality (IQ)

A unique qualification assay that calculates the ratio of large, intact, and/or structured RNA to small, degraded RNA.

Protein quantification

Qubit protein assays rapidly measure protein content across two different concentration ranges, both broader than traditional Bradford protein quantification assays.

Ion Sphere Quality Control Kit

Specially designed to assess enriched Ion Sphere Particles (ISPs) prior to sequencing on the Ion Personal Genome Machine (PGM) or Ion GeneStudio System.

Custom MyQubit assays (Qubit 4 only)

Create your own quantification assays for the Qubit 4 Fluorometer.

Fluorometer mode and MyQubit custom assays

In Fluorometer mode, the Qubit Fluorometer can be used as a mini-fluorometer. Rather than performing an assay calibration and calculating results based on an algorithm, the instrument generates and displays raw fluorescence unit (RFU) values for each sample.

 

In Fluorometer mode, you can choose either the blue or red LED as the excitation source. If you choose blue excitation, the instrument reads fluorescence values in both the green and far-red emission channels. If you choose red, emission is read in the far-red channel only.

Fluorometer mode

To access Fluorometer mode, press Fluorometer on the home screen. Choose whether you want to use the blue (470 nm) or red (635 nm) excitation source. Once you insert your sample and select Read tube, your results appear on the screen in RFUs.

MyQubit assays

Data gathered in Qubit 4 Fluorometer mode can be used to design your own assay using the MyQubit assay design tool. This tool lets you create new custom assays for the Qubit 4 Fluorometer in minutes. Simply enter your assay parameters into the online tool, and then save and upload the .qbt file to your Qubit Fluorometer using a USB drive.

 

Preconfigured MyQubit assays to quantify cholesterol, galactose, glucose, glutamic acid, peroxide, or sucrose are available. You can download these .qbt files to your computer and then upload them to your Qubit 4 Fluorometer using a USB drive. These are examples of the kinds of MyQubit assays you can create on your own.

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