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
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|
Figure 1. Workflow for the Quant-iT™ assays using the Qubit® Fluorometer.
Selectivity for DNA or RNA
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
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
Figure 4. Comparison of sample concentration ranges for the Quant-iT™ assays using the Qubit® Fluorometer and UV absorbance measurements using the NanoDrop® spectrophotometer.
- Glasel JA (1995) Biotechniques 18:62–63.
- Huberman JA (1995) Biotechniques 18:636.
- Manchester KL (1995) Biotechniques 19:208–210.
- Manchester KL (1996) Biotechniques 20:968–970.
- Singer VL, Jones LJ, Yue ST et al. (1997) Anal Biochem 249:228–238.
- Jones LJ, Yue ST, Cheung CY et al. (1998) Anal Biochem 265:368–374.
- LePecq JB (1966) Anal Biochem 17:100–107.
- Kapuscinski J (1995) Biotech Histochem 70:220–233.