Comparison of the Qubit® Quantitation Platform with Spectrophotometry
Detection and quantitation of nucleic acids are vital to many biological studies. Historically, DNA and RNA have been quantitated using spectrophotometry to measure absorbance at 260 nm. Although this method is most commonly used, it can be unreliable and inaccurate [1–4]. UV absorbance measurements are not selective and cannot distinguish DNA, RNA, or protein. Measurements are easily affected by other contaminants(e.g., free nucleotides, salts, and organic compounds) and variations in base composition. In addition, the sensitivity of spectrophotometry is often inadequate, prohibiting quantitation of DNA and RNA at low concentrations.
In light of these drawbacks, the use of fluorescent dyes to quantitate nucleic acids has become a common alternative [5–8]. Fluorescence-based quantitation is more sensitive and is often specific for the nucleic acid of interest. In particular, the Qubit® Quantitation Platform provides a more selective, sensitive, and accurate method for quantitating nucleic acids than UV absorbance measurements, including those obtained with the NanoDrop® ND-1000 Spectrophotometer.
NOTE: This article was published in March 2010. Since that time, we have launched the next generation instrument, the Qubit® 2.0 Fluorometer which quantitates DNA, RNA, and protein with unprecedented accuracy, sensitivity, and simplicity. Additionally, the associated Quant-iT™ Assay Kits have been rebranded as Qubit® Assay Kits.
The Qubit® Quantitation Platform: Fast and Easy To Use
The Qubit® Quantitation Platform is the combination of a user-friendly fluorometer with highly sensitive fluorescence-based quantitation assays. The Qubit® Fluorometer is a small, economical instrument designed to work seamlessly with Quant-iT™ Assay Kits for routine DNA, RNA, and protein quantitation (Table 1). All settings and calculations are performed for you. The system is simple, fast, and easy to use, yet consistently produces accurate results so that you can be confident moving forward with subsequent applications. Each Quant-iT™ assay kit is highly specific for a single analyte, and all are more sensitive than absorbance-based measurements. Small sample volumes of only 1–20 μL are required, which means less sample used for quantitation and more sample available for analysis.
Table 1. Quant-iT™ Assay Kits For Use With The Qubit® Fluorometer.
|Kit||Sample Starting Concentration Range|
|Quant-iT™ dsDNA HS Assay||10 pg/μL–100 ng/μL|
|Quant-iT™ dsDNA BR Assay||100 pg/μL–1 μg/μL|
|Quant-iT™ ssDNA Assay||50 pg/μL–200 ng/μL|
|Quant-iT™ RNA Assay||250 pg/μL–100 ng/μL|
|Quant-iT™ RNA BR Assay||1 ng/μL–1 μg/μL|
|Quant-iT™ Protein Assay||12.5 μg/mL–5 mg/mL|
The Quant-iT™ assays for use with the Qubit® Fluorometer are all performed using the same general protocol. A simple mix-and-read format is used, with incubation times of only 2 minutes required for DNA and RNA assays (Figure 1).
Figure 1. Workflow for the Quant-iT™ assays using the Qubit® Fluorometer.
Selectivity for DNA or RNA
The most significant difference between using the Qubit® Quantitation Platform and UV absorbance to measure nucleic acid concentrations is the selectivity of the Quant-iT™ assays, which are very specific for the molecule of interest and provide much more accurate information than UV absorbance.
With UV analysis, results for samples containing both DNA and RNA are nondiscriminatory—you cannot distinguish one from the other. In contrast, the Qubit® Quantitation Platform enables accurate measurement of both DNA and RNA from the same sample, using a combination of kits (Figure 2). In this experiment, the DNA concentration of a sample containing equal parts DNA and RNA was measured to within 2% of the actual concentration using the Quant-iT™ DNA BR Assay Kit with the Qubit® Fluorometer. Furthermore, in a sample containing a 10-fold excess of RNA over DNA, the concentration determined in the DNA assay was only 7% higher than the actual concentration. DNA and RNA in samples such as these could not be accurately measured using UV absorbance on the NanoDrop® spectrophotometer, increasing the likelihood of error in subsequent applications.
Figure 2. Selectivity of the Quant-iT™ assays compared to UV spectrophotometry.Triplicate samples containing lambda DNA (10 ng/μL) and varying amounts of E. coli ribosomal RNA (0 to 100 ng/μL) were assayed using Quant-iT™ DNA BR and Quant-iT™ RNA BR assays on the Qubit® 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 PerkinElmer 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 PerkinElmer Lambda 35 Spectrophotometer. The concentrations of the stock solutions were then calculated and used for all subsequent dilutions. The data indicate that with UV analysis, results for samples containing both DNA and RNA are nondiscriminatory.
Good Accuracy and Precision, Even At Low Concentrations
The Qubit® Quantitation Platform generates more accurate and precise results across a lower concentration range than those obtained by UV absorbance measurements on the NanoDrop® spectrophotometer. Using the Quant-iT™ dsDNA HS Assay Kit, the Qubit® Fluorometer quantitates DNA in samples with concentrations as low as 10 pg/μL to within 12% of the actual concentration (Figure 3A). In contrast, the concentrations in these same samples were overestimated 46-fold when measured with the NanoDrop® spectrophotometer. Likewise, samples containing 10 ng/μL of DNA (the reported low end of quantitation for the NanoDrop® spectrophotometer is 2 ng/μL) are accurately read to within <1% of the actual concentration using the Qubit® Fluorometer and 5% using the NanoDrop® spectrophotometer. In addition, the variation of replicates (% CV) for all samples containing at least 0.5 ng/μL of DNA was ≤1% using the Qubit® Fluorometer (Figure 3B). Only samples containing 4 ng/μL of DNA or higher resulted in % CVs lower than 9% using the NanoDrop® spectrophotometer.
Figure 3. 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™ dsDNA HS Assay on the Qubit® 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 (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.
Broad Range and High Sensitivity
The Quant-iT™ assays used on the Qubit® Fluorometer are more sensitive than UV absorbance measurements, and because the assays can tolerate 1–20 μL of sample, the effective range of the assays can be increased (Figure 4). Together, the Quant-iT™ DNA HS and BR assays cover a sample concentration range of 10 pg/μL to 1 μg/μL DNA. Similarly, the Quant-iT™ RNA and RNA BR assays cover a sample concentration range of 250 pg/μL to 1 μg/μL. In contrast, the NanoDrop® spectrophotometer covers a sample concentration range of 2 ng/μL to 15 μg/μL, as reported by the manufacturer.
Figure 4. Comparison of sample concentration ranges for the Quant-iT™ assays using the Qubit® Fluorometer and UV absorbance measurements using the NanoDrop® spectrophotometer.
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