Qubit 4 Fluorometer is the instrument of choice when accuracy matters most

Trusted when accuracy matters

Invitrogen 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 Invitrogen Qubit Fluorometer is the instrument of choice when accuracy matters most.


Testimonials

 

Quick and easy with excellent repeatability; more reliable than spectrophotometry and more confidence in results.

— Kevin Barr, University of Western Ontario

It gives me the possibility to measure very diluted samples. Good, quick, and easy.

— Sylvia Rodriguez, Institute for Research in Biomedicine (IRB Barcelona)


Qubit Fluorometer vs. Quantus Fluorometer

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 Invitrogen 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.

  bar graph showing Qubit assay performance compared to QuantFluor assays on the Quantus Fluorometer for low-concentration DNA samples  

Figure 1. Assay sensitivity.
Comparable results are achieved at 0.5 ng/mL for both the Invitrogen 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

Invitrogen 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.

  bar graph showing Qubit assay performance compared to QuantFluor assays on the Quantus Fluorometer for DNA-contaminated RNA samples  

Figure 2. Accuracy in the presence of DNA.
The Invitrogen 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
dsDNA Yes Yes
RNA Yes Yes§ 
microRNA  Yes No 
Protein  Yes No 
ssDNA or oligos Yes Yes
‡New assays including those for cholesterol and glucose can be added to the Invitrogen Qubit 2.0, 3.0, and 4 Fluorometers.  
§QuantiFluor RNA assay for the Quantus Fluorometer also detects DNA. 

Qubit fluorescence quantitation vs. UV-absorbance quantitation

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 Invitrogen 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.

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 Invitrogen 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 Invitrogen 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.

References

  1. Glasel JA (1995) Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques 18:62-63.
  2. Huberman JA (1995) Importance of measuring nucleic acid absorbance at 240 nm as well as at 260 and 280 nm. Biotechniques 18:636.
  3. Manchester KL (1995) Value of A260/A280 ratios for measurement of purity of nucleic acids. Biotechniques 19:208–210.
  4. Manchester KL (1996) Use of UV methods for measurement of protein and nucleic acid concentrations.Biotechniques 20:968–970.

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