Trusted when accuracy matters
Qubit® instruments and assays have a proven track record, with over 3,000 citations and hundreds of testimonials. With high sensitivity for DNA, high specificity for RNA, and a broad range of available assays, the Qubit® Fluorometer is the instrument of choice when accuracy matters most
The Qubit® Fluorometer is an analytical instrument for DNA, RNA, and protein quantification with a small benchtop footprint and an intuitive user interface. The instrument works with Qubit® assay kits to enable greater sensitivity and accuracy than UV absorbance measurements, and is ideal for applications including cloning, sequencing, transfection, qPCR, and protein assays. Here we compare accuracy and specificity data for the Qubit® Fluorometer and assays with the Quantus™ Fluorometer and QuantiFluor® assays from Promega.
DNA assay sensitivity
Promega claims that dsDNA assays on the Quantus™ Fluorometer are 10 times more sensitive than Qubit® Fluorometer dsDNA assays,* but assay precision is compromised for both instruments at concentrations below 0.5 ng/mL (Figure 1). The Qubit® Fluorometer gives an out-of-range message when the calculated value exceeds a CV of 20%, providing you with confidence in the results. Table 1 summarizes these results along with other attributes of each instrument.
Figure 1. Assay sensitivity. Comparable results are achieved at 0.5 ng/mL for both the Qubit® dsDNA HS assay and the QuantiFluor® dsDNA assay, but the coefficient of variation (CV) for the QuantiFluor® assay is 112% at 0.1 ng/mL. The Qubit® fluorometer reports an out-of-range message at levels >20% CV; the Quantus™ Fluorometer does not.
Table 1. Comparison of dsDNA quantification data and other instrument features.
|Qubit® Fluorometer and assays||Quantus™ Fluorometer and QuantiFluor® assays|
|Sensitivity (dsDNA)||0.5 ng/mL (10% CV)||0.1 ng/mL (112% CV)|
|Dynamic range† (dsDNA)||0.5 ng/mL to 5 µg/mL||0.1 ng/mL to 1 µg/mL|
|Detection limit||Yes—out-of-range message at low and high ends of assays||No—gives concentration values for any sample with fluorescence signal above background|
|Display||Large, intuitive touch screen||Push-button navigation|
|Data storage||1,000 samples||20 samples|
|Data transfer||USB thumb drive or cable||Indirect—need to download software onto computer|
|† Combined range of Qubit® Broad Range and High Sensitivity assays.|
RNA quantification accuracy in the presence of DNA
Qubit® RNA assays are designed to be specific and accurate for RNA even in the presence of a 1:1 mixture of RNA and DNA, whereas the QuantiFluor® RNA assay on the Quantus™ Fluorometer cannot distinguish RNA from DNA below 3 µg/mL (Figure 2). This is critical for limited, low-concentration samples that may also contain DNA.
Figure 2. Accuracy in the presence of DNA. The Qubit® RNA BR (broad range) assay demonstrates high specificity, even at low concentrations and in the presence of DNA. A significant amount of DNA is detected as RNA with the QuantiFluor® assay.
The Qubit® Fluorometer has more compatible assays
The Qubit® Fluorometer supports assays for high-sensitivity or broad-range dsDNA and RNA quantitation (Table 2). Assays for oligos, ssDNA, protein, and microRNA quantification are also available.
Table 2. Assays available for each fluorometer.
|Assay‡||Qubit® assays for the Qubit® Fluorometer||QuantiFluor® assays for the Quantus™ Fluorometer|
|ssDNA or oligos||Yes||Yes|
|‡New assays including those for cholesterol and glucose can be added to the Qubit® 2.0 and 3.0 Fluorometers.
§QuantiFluor® RNA assay for the Quantus™ Fluorometer also detects DNA.
Detection and quantification of nucleic acids are vital to many biological studies. Historically, DNA and RNA have been quantified using spectrophotometry to measure absorbance at 260 nm. Although this method is very common, in some applications it can be inaccurate [1–4].
The Qubit® fluorescence and UV-absorbance microvolume instruments may be used together to determine RNA or DNA concentration—the Qubit® Fluorometer for accurate quantification of the target molecule and the UV-absorbance spectrophotometer to indicate contaminants. We have compared the Qubit® Fluorometer to a popular microvolume spectrophotometer. The major differences are listed in Table 3, illustrated in Figures 3–5, and demonstrated in this video.
See how fluorescence quantitation works compared to UV-absorbance quantitation
Table 3. Quantification method comparison.
|Qubit® Fluorometer||UV-absorbance microvolume spectrophotometer|
|Quantification method||Fluorescence-based dyes that bind specifically to DNA, RNA, or protein||UV absorbance measurements (measures absorbance at 260 nm and 260 nm/280 nm ratio)|
|Selectivity for DNA or RNA (Figure 3)||Accurately measure both DNA and RNA in the same sample||Results for samples containing both DNA and RNA are nondiscriminatory—you cannot distinguish one from the other|
|Accuracy and precision at low concentrations (Figure 4)||Accurately quantifies DNA in samples with concentrations as low as 10 pg/μL||Not recommended for concentrations under 2 ng/μL; variation for sample concentrations <10 ng/μL is often high|
|Sensitivity and range (Figure 5)||The effective range covers a sample concentration range of 10 pg/μL to 1 μg/μL DNA||Covers a sample concentration range of 2 ng/μL to 15 μg/μL; uses 0.5–2 μL of sample|
|Can indicate contamination||No||Gives peaks revealing the presence of contaminants|
|Figure 3. Selectivity of the Qubit® assays compared to UV spectroscopy. Triplicate samples containing lambda DNA (10 ng/μL) and varying amounts of ribosomal E. coli RNA (0–100 ng/μL) were assayed using Qubit® DNA BR and Qubit® RNA BR assays on the Qubit® Fluorometer according to kit protocols. The same samples were subsequently measured in triplicate using a microvolume spectrophotometer, and single measurements were made using a cuvette-based spectrophotometer. The concentrations indicated are the concentrations of DNA and RNA in the starting samples, before dilution in the Qubit® assay tubes. The red and orange trendlines indicate the actual concentrations of DNA and RNA, respectively, in the starting samples. The actual concentration of nucleic acid was set by diluting pure, concentrated solutions of DNA and RNA to an optical density of 1.0 at 260 nm using the cuvette-based spectrophotometer. The concentrations of the stock solutions were then calculated and used for all subsequent dilutions. With UV analysis, results for samples containing both DNA and RNA are nondiscriminatory—you cannot distinguish one from the other..|
|Figure 4. Accuracy and precision of Qubit® fluorometric quantification. Ten replicates of lambda DNA at concentrations from 0.01 to 10 ng/μL were assayed using the Quant-iT® DNA HS Assay on the Qubit® Fluorometer according to the standard kit protocol. The same concentrations of DNA were measured in 10 replicates using a UV-absorbance microvolume spectrophotometer, and results were compared for both accuracy (A) and precision (B). Accuracy was defined as the average deviation from the known concentration. The concentrations indicated are the concentrations of DNA in the starting samples, before dilution in the Qubit® assay tubes.|
|Figure 5. Comparison of sample concentration ranges for the Qubit® assays using the Qubit® Fluorometer and UV absorbance measurements using a microvolume spectrophotometer.|
- Glasel JA (1995) Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques 18:62-63.
- Huberman JA (1995) Importance of measuring nucleic acid absorbance at 240 nm as well as at 260 and 280 nm. Biotechniques 18:636.
- Manchester KL (1995) Value of A260/A280 ratios for measurement of purity of nucleic acids. Biotechniques 19:208–210.
- Manchester KL (1996) Use of UV methods for measurement of protein and nucleic acid concentrations.Biotechniques 20:968–970.
For Research Use Only. Not for use in diagnostic procedures.