Qubit® Fluorometer vs. NanoDrop® Spectrophotometer

Detection and quantitation 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 most commonly used, it can be unreliable and inaccurate [1–4].

We have compared the Qubit® 2.0 Fluorometer to the NanoDrop® ND-1000 spectrophotometer. The major differences are listed in the table below.

    Qubit Fluorometer   NanoDrop ND-1000   The Data
Quantitation Method Fluorescence-based dyes that bind specifically to DNA, RNA or protein UV absorbance measurements (measures 260 nm/280 nm ratio)  
Selectivity for DNA or RNA 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 See Figure 1
Accuracy and Precision at Low Concentrations Accurately quantitates DNA in samples with concentrations as low as 10 pg/μL Significantly overestimates the measurement with the same sample—variation is often high See Figure 2
Sensitivity and Range Tolerates 1–20 μL of sample. 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 See Figure 3

Selectivity for DNA or RNA


Figure 1. 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® 2.0 Fluorometer according to kit protocols. The same samples were subsequently measured in triplicate using a NanoDrop® ND-1000 Spectrophotometer, and single measurements were made using a Perkin Elmer Lambda 35 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 a Perkin Elmer Lambda 35 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.

Accuracy and Precision at Low Concentrations


Figure 2. Accuracy and precision of the Qubit® Quantitation Platform. 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® 2.0 Fluorometer according to the standard kit protocol. The same concentrations of DNA were measured in 10 replicates using a NanoDrop® ND-1000 Spectrophotometer, and results were compared for both accuracy and precision. Each bar represents the average of 10 replicates. Error bars represent the standard deviations of the 10 replicates. The concentrations indicated are the concentrations of DNA in the starting samples, before dilution in the Qubit® assay tubes.

Sensitivity and Range

 Figure 3. Comparison of sample concentration ranges for the Qubit® assays using the Qubit® 2.0 Fluorometer and UV absorbance measurements using the NanoDrop® spectrophotometer.
Learn more differences between the two instruments from Common Practices for Routine RNA Handling in the "Quality Control of RNA" section (see page 21).

References

Qubit® 2.0 Fluorometer: A Researcher's Story


Researcher Jason Carriere of the University of Oregon discusses the benefits of having Qubit® 2.0 in the lab.