White Paper Absorbance Or Fluorescence: Which Is the Best
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WHITE PAPER No. 40 Absorbance or Fluorescence: Which Is the Best Way to Quantify Nucleic Acids? Natascha Weiß1, Martin Armbrecht1 ¹Eppendorf AG, Hamburg, Germany Executive Summary When performing molecular experiments on nucleic acids, it is a basic requirement to determine the concentration as well as the quality of the sample. The standard method which serves this purpose is UV-Vis spectrophotometry – but is it always the right choice? In the present White Paper, the advantages and disadvantages of this technique will be described and it will be compared with nucleic acid quantification via fluorescence. In this context, it will be discussed which situations warrant the use of which one of the two methods. The decision will generally depend on the condition of the sample as well as on the requirements of downstream applications. Since the advantages of both methods complement each other well, it is most practical to have the ability to perform both methods on a single instrument in a flexible manner. Introduction Experiments involving nucleic acids are a mainstay in any evaluated by measuring the sample at additional wave molecular laboratory. DNA and RNA are isolated from micro- lengths (230 nm, 280 nm) and calculating the purity ratios, organisms and from cells of higher order organisms in order i.e. the ratios of the values obtained at 260/230 nm and at to be employed in a broad variety of processing steps and 260/280 nm, respectively. In this way, it will be evident analyses. It is crucial for any type of downstream applica- whether cellular debris or remainders of reagent used during tion that a defined amount of nucleic acid is used and that purification such as proteins, sugar molecules, certain salts the sample is free from contaminations that may impact the or phenols, are present in the solution, as these will generate experiments. To this end, subsequent to the isolation of the a different absorbance spectrum from nucleic acids (figure 1). nucleic acid, its concentration and, if required, its purity are In addition, turbidity measurements, frequently conducted at determined on a routine basis. 320 nm, are capable of detecting particles within the sample [1]. A scan which covers the entire UV spectrum will provide One tried and true method is the quantification of DNA/RNA even more information on impurities, and particularly on solutions by measuring absorbance at a wavelength of 260 nm possible errors which may have occurred during the course in a UV-Vis (spectro-)photometer. Sample purity may be of the measurement [2]. WHITE PAPER I No. 40 I Page 2 230 260 Layout280 Nucleic acids Proteins 1.6 1.4 1.2 1 0.8 0.6 Absorbance Sugars, salts and 0.4 organic solvents 0.2 0 240 260 280 300 320 340 Wavelength [nm] Figure 1: Spectra obtained from nucleic acids and possible contaminants The major advantage of this direct method is the fact that it is suffi cient to even fi ll a cuvette. Microvolume systems, which very simple and quick to perform. With the right equipment require sample volumes of only 1 – 2 µL, may off er a solu- (micro-volume instrument or micro-volume cuvette), it is tion. It is, however, important to keep in mind that, due to the possible to measure very small volumes (in the range of shorter path lengths of these systems (compared to a path 1 µL) and thus save sample material. In addition, as elabo- length of 10 mm in common cuvettes), their detection limits rated above, this method is capable of determining sample are correspondingly higher. For this reason, considerably purity. higher sample concentrations are required in order to obtain accurate and reproducible measurement values. This method, however, comes with certain drawbacks: on the one hand, with a detection limit in the range of approximately Furthermore, all nucleic acids generate the same absorbance 1 µg/mL dsDNA (based on the common path length of spectrum (fi gure 2), thus potentially leading to an overestima- 10 mm), it is not very sensitive. Therefore, in order to guaran- tion of the concentration of the DNA at hand in cases where tee accurate quantifi cation, samples may need to be concen- RNA is present alongside dsDNA [3]. Contaminations are trated prior to measurement, which, in turn, entails additional also frequently capable of absorbing light at 260 nm and thus eff ort and which also frequently leads to considerable sample contribute to elevated readings (fi gure 1). loss. In extreme cases, the amount of sample will not be Hypochromic shift of different nucleic acids 0.4 ssDNA RNA dsDNA 0.3 0.2 Absorbance 0.1 0 220 230 240 250 260 270 280 290 300 310 320 Figure 2: Absorbance spectra of diff erent Wavelength types of nucleic acids. WHITE PAPER I No. 40 I Page 3 • Detection basics Fluorescence measurements – the alternative? Fluoreszenz-‐PrinzipCommon alternatives employed in the quantification of Quantification of nucleic acids via fluorescence is based on nucleic acids include measurements of fluorescence, qPCR the use of fluorescent dyes which bind to the molecule of and capillary electrophoresis systems that are chip-based. interest. Only the complex consisting of nucleic acid and dye The two latter processes are mainly used for specific tasks is excited by light of a specific wavelength (dependent on (e.g. qPCR: exact quantification of specific target sequences; the dye) and will subsequently emit light of a slightly longer capillary electrophoresis: exact determination of fragment wavelength (figure 3). The intensity of the fluorescent signal size and integrity) as both methods are relatively costly as is dependent on the amount of nucleic acid. For excitation well as time-consuming. and detection, a plate reader with fluorescence function, a fluorimeter3-4: or Lichtemissiona photometer with a fluorescence (EM)/ module is required.Fluoreszenz 1 A 4 B Excitation atomic Emission EX EM electron nucleus E Target 3 Fluorophore 2 Figure 3: A) Principle of fluorescence measurements of nucleic acids. Wavelength [nm] B) The light used to excite the fluorophore (EX) has a shorter wavelength than the fluorescent light emitted (EM). 1 – Elektron wird durch Lichtabsorption auf ein höheren Energielevel gebracht (Absorptionsmessung) undOne major advantage of this method is its high sensitivity, Detection of nucleic acids via fluorescence is, however, an which is 1000-fold higher for the fluorescent dye PicoGreen® indirect method that necessitates the generation of a stan- 2 – Elektron springt zurückas compared auf seinen to absorbance ursprünglichen measurements. In this way, Energielevel even dard curve prior to the actual measurement. Since samples 3 – Elektron kehrt nicht direktsamples of auf very low seinen concentrations ursprünglichen can be quantified ac- Energieand standards level must besondern prepared with zunächst the fluorescent dye, zu einem Zwischenlevel /curately 4 – [4].Elektron A further positive springt aspect is vonthe fact demthat this Zwischenlevelde- this method is slightly auf more seinen cumbersome than UV-Vis spec- tection method requires only very small amounts of sample. trophotometry (figure 4). Furthermore, this method does not ursprünglichen EnergielevelFluorescent (Fluoreszenz-Strahlung dyes bind in a highly specific manner wird– possible frei) allow one to draw any conclusions regarding the purity of contaminations such as salt or protein will therefore not lead the sample. to artificially elevated readings, which equals higher accu- racy. Certain variants are available which bind specifically 1 1 to one type of nucleic acid. This is the case for dyes which detect dsDNA in particular, thus practically eliminating the impact of possible ssDNA or RNA on the result. WHITE PAPER I No. 40 I Page 4 Workfl ow fl uorescence measurements Sample preparation B > Add fl uorescence reagent to blank, standards and samples > Incubate A Generate standard curve B Determine sample concentration > Measure blank and standards > Measure samples > Choose regression mode > Calculate concentration Figure 4: Workfl ow for the quantifi cation of nucleic acids via fl uorescence measurements. The decision which of the two methods is to be used for ideally suited to the verifi cation of sample purity. Fluores- nucleic acid quantifi cation depends on a number of fac- cence measurements are benefi cial in cases where the exact tors including, on the one hand, the amount and quality of quantifi cation of samples of low concentration, or of samples the sample, and on the other hand, the requirements of the containing impurities, is of the essence. downstream application. It is crucial to weigh the impor- tance of accurate determination of the sample concentration It is frequently a good idea to combine both methods, thus against the potential impact of impurities on subsequent allowing the respective benefi ts to complement each other. applications. In general, absorbance measurement is the The advantages of both detection methods are summarized method of choice for samples of medium to high concen- in table 1. tration, as well as high purity. Alternatively, this method is Table 1: Advantages of absorbance measurements and fl uorescence measurements, respectively Quick and easy as this is a direct method of measurement Absorbance Non-hazardous and very stable as no chemicals are required Provides information on sample purity High sensitivity; therefore ideally suited to the analysis of samples of low Fluorescence concentration and saving of valuable sample material High accuracy due to high specifi city for the target molecule WHITE PAPER I No. 40 I Page 5 Solutions by Eppendorf The Eppendorf BioSpectrometer® fluorescence is an instru- across a broad measurement range (figure 6). In addition, ment that enables both absorbance measurements as well sample purity is easily determined via absorbance measure- as fluorescence measurements (figure 5). This combination ments, whereas the fluorescence method is highly specific. allows accurate quantification of nucleic acid solutions A B Figure 5: A) BioSpectrometer fluorescence B) Directions of the light paths within the instrument for the respective methods, absorbance and fluorescence, are indicated.