Nucleic Acid Quantitation
Understand your sample’s purity and concentration
For scientists working with DNA, RNA, or proteins, sample quality often plays a critical role in determining the success or failure of an experiment.
The Thermo Scientific NanoDrop One UV-Vis Spectrophotometer and the Invitrogen Qubit 4 Fluorometer, when used together, provide the ability to obtain the most complete information about the concentration and quality of your DNA, RNA, or protein sample. Knowing the precise concentration and purity of your sample early helps you prevent costly troubleshooting and rework downstream.
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Quantitation—Comparing Qubit fluorometer to the Nanodrop One UV/Vis absorbance
The best of both worlds — the combination of the NanoDrop One UV-Vis Spectrophotometer and the Qubit 4 Fluorometer enables accurate, easy, and fast quantitation of biomolecules.
Which instrument do I need?
For workflows that involve nucleic acids, determining the concentration (quantity) and purity (quality) of the sample is often a necessary step. Ultimately, the more you know about your sample up front, the lower your potential risk of experimental failure later.
While both the NanoDrop One Spectrophotometer and Qubit 4 Fluorometer offer accurate measurement of DNA, RNA, and protein samples, each also has unique advantages.
Cited in more than 30,000 publications, NanoDrop UV-Vis microvolume spectrophotometers fundamentally changed the way scientists evaluate nucleic acid and protein samples by eliminating the need for large sample volumes.
The next-generation NanoDrop One microvolume spectrophotometer utilizes Thermo Scientific Acclaro sample intelligence technology, which identifies contaminants, provides corrected concentration values, and helps you assess the quality of your sample before you use it in downstream applications.
The Qubit 4 Fluorometer is a cutting-edge benchtop device that accurately measures DNA, RNA, protein quantity and RNA integrity, and quality using the highly sensitive Invitrogen Qubit assays.
In conjunction with state-of the-art, highly optimized algorithms and a seamless user interface, the Qubit 4 Fluorometer employs fluorescent dyes that only produce signal when bound to the target of interest, thereby minimizing the effects of contaminants— including degraded DNA or RNA—on your results.
|NanoDrop One Spectrophotometer||Qubit 4 Fluorometer|
Fast and simple—allows direct measurement of purified samples with no dilutions required
Sensitive and specific—measures nucleic acid concentration accurately whether dsDNA, ssDNA, total RNA, or microRNA, even in the presence of contaminants. With the RNA IQ assay, Qubit 4 is able to measure intact or large RNA from degraded or small RNA in 5 minutes.
|Workflow||Pipette sample onto pedestal; measure||Mix sample with reagents, incubate for 2-5 minutes, read.|
|Applications||Routine assessment of quantity and quality (purity) of DNA, RNA, and proteins||
Use with very dilute or difficult-to-process samples, when higher sensitivity is required or when nucleic acid contaminants are suspected (e.g., DNA in RNA; presence of dNTPs). Also use for rapid assessment of RNA integrity and quality.
|dsDNA quantification range (ng/μL)||0.2*– 27,500||0.01–1,000|
|Sample quality assessment (purity)||
Acclaro sample intelligence technology identifies presence of contaminants such as protein or guanidine; provides corrected analyte concentration value
dsDNA sample contaminated with protein. The absorbance contribution from the protein (orange) is subtracted from the original result (blue) to obtain the corrected dsDNA concentration (green).
Unique fluorescent dyes enable differentiation between sample types, including nucleic acids (e.g., DNA vs. RNA), without any sample pretreatment.
The Qubit dsDNA HS Assay is selective for dsDNA, even in the presence of an equal mass of RNA.
* dsDNA quantitation range for NanoDrop One is 0.2 ng/μL with cuvette and 2.0 ng/μL without.
For samples that are 100% pure and made of a single nucleic acid type (e.g., dsDNA), the results obtained on the NanoDrop One Spectrophotometer and the Qubit 4 Fluorometer would be expected to be identical for samples within the instruments’ dynamic ranges. However, few samples are 100% pure.
For most labs working with nucleic acids, it is highly advantageous to have the speed and flexibility of the NanoDrop One Spectrophotometer along with the specificity and sensitivity of the Qubit 4 Fluorometer. Together, the two elegantly designed instruments provide you with precise quantification and intelligent quality assessment of your biomolecule of interest, and help save your lab significant time and money by helping to prevent downstream experimental failures.
Quantitating nucleic acids: downstream applications guide
|Application||NanoDrop One Spectrophotometer||Qubit 4 Fluorometer|
|General PCR||Routine PCR applications have become robust over the years. Using UV to quantify the template is sufficient.||Good for routine samples, preferred if sample is prone to high amounts of contaminants that absorb at 260 nm (A260) (dNTPs, oligos, RNA, etc.).|
|Quantitative PCR||UV allows the researcher to determine the presence of contaminants that may affect the polymerase reaction (e.g., phenol).||Reliable qPCR results require highly accurate template concentrations. The specificity and sensitivity of Qubit assays are well suited for this task.|
|Sequencing||Using UV to quantify the template is sufficient, except if the sample is prone to high amounts of contaminants that absorb at 260 nm (A260) (e.g., RNA in DNA samples).||If samples are prone to contaminants that absorb at 260 nm (A260), the high specificity of a Qubit assay is advantageous.|
|Next-generation sequencing||UV for sample evaluation during the library construction process. Allows the researcher to determine presence of contaminants that may affect different stages of the workflow.||Reliable NGS results require highly accurate template concentrations. The specificity and sensitivity of Qubit assays are well suited for this task.|
|RT-qPCR||Standard RNA extractions are less prone to DNA contamination, so UV measurement can be used to normalize the amount of RNA that goes into the reaction.||The specificity and sensitivity of Qubit assays are good for routine samples, and desirable if the sample is prone to DNA contamination or degraded RNA (dNTPs and NTPs).|
|QC of dye-labeled microarray probes||Enables simultaneous measurement of the amount of dye and probe.||NA||Not recommended for QC of dye-labeled microarray probes, but Qubit assays can be used to accurately measure microarray probe concentration.|
|Genotyping / mutation detection||A260 measurement can overstate concentrations due to mixed contaminants.||Samples often contain contaminants that absorb at 260 nm (A260), making the specificity of the Qubit assay desirable.|
|NanoDrop One Spectrophotometer||Qubit 4 Fluorometer|
Selection of preprogrammed applications
Total protein concentration for three samples shown as red squares on a BCA assay standard curve (top panel); four different purified protein samples were measured using the Protein A280 application (bottom panel).
|Qubit assays exhibit minimal protein-to-protein variability and are resistant to most contaminants, including salts, reducing agents (DTT, β-mercaptoethanol), DNA, and amino acids, but not detergents.
Low protein-to-protein variation in the Qubit protein assay. Solutions of the following proteins were assayed: bovine serum albumin (BSA), chicken egg ovalbumin, chicken egg lysozyme, bovine milk β-casein, equine myoglobin, bovine milk α-casein, porcine pepsin, mouse immunoglobulin (IgG), and calf thymus histone.
Use fluorometers to quantify, detect and monitor analytes and their reactions by measuring the intensity of the fluorescent signal from dyes attached to biological molecules as well as naturally fluorescent molecules based on signature excitation (Ex) and emission (Em) wavelengths.
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NanoDrop One Spectrophotometer and Qubit 4 Fluorometer
For scientists working with DNA, RNA, or proteins, sample quality can play a critical role in determining the success or failure of an experiment. Together, the NanoDrop One UV-Vis spectrophotometer and the Qubit 4 fluorometer provide your lab with the ability to obtain the most complete information about the concentration and quality from your DNA, RNA, or protein sample.
To request a quote or demo for one or both devices, complete our form and a technical specialist will contact you to make arrangements.
For Research Use Only. Not for use in diagnostic procedures.