Fluorometer: high detection sensitivity, wide detection range, short detection time, specificity
DNA and RNA quantification, commonly referred to as nucleic acid quantification, is often used to determine the average concentration of DNA or RNA in a sample prior to downstream experiments. Sample purity is also an important consideration in accurately calculating the amount of DNA or RNA in a sample. There are two commonly used optical techniques to quantify nucleic acids:UV-visible measurements and fluorescence measurements. Choosing the right technique for your sample and workflow can accurately quantify RNA or DNA and can save a great deal of time and money by helping to prevent downstream experimental failures.
There are two main types of equipment on the market today: one is an Ultra Trace UV Spectrophotometer and the other is a Fluorometer. Ultra Trace UV Spectrophotometer is a simple method that does not require reagents and can be tested with a single drop. However, this method is not very accurate. Fluorometer is specific. It can specifically detect different targets and needs to be used with specific kits. Our Innova portable fluorometer models are PFM-01X2 and PFM-08X1.
| Photometry (UV-Vis) | Fluorescence |
| How is the optical signal generated? | The photometric measurement of nucleic acids is based on the intrinsic absorptivity properties of nucleic acids (DNA and RNA). When an absorption spectrum is measured, nucleic acids absorb light with a characteristic peak at 260 nm. | The fluorometric measurement of nucleic acids is based upon the use of fluorogenic dyes that bind selectively to DNA or RNA. |
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Typical RNA/DNA absorbance spectrum | 
Fluorescent dyes selectively bind to DNA, RNA or protein. Dyes only emit signal when bound to the target. |
How is the optical signal measured?
| The signal is measured byT spectrophotometers or spectrometers. The attenuation in the light that reaches the detector after passing through the sample is measured in relation to the incident light and expressed as absorbance values of the sample in the solution.
| The signal is measured by fluorometers.
Sample is excited with filtered light(at the excitation wavelength), and the emitted light (at the emission wavelength) is recorded by a detector. |
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| Wavelength separation can take place before or after the light has passed the sample, and the optical light path can be horizontal or vertical. | Wavelength separation can take place in various ways (for example with filters or with monochromators) |
| What are the advantages? | 1.It is simple—no sample preparation, dyes, or standards are required
2.Can provide direct measurements of purity ratios—A260/280 and A260/230
3.Can provide information on contaminants—can identify non-nucleic acid contamination in samples (proteins, phenol, guanidine salts) as well as mammalian DNA/RNA contamination and provide corrected concentrations. | 1.It is specific—performed measurement is selective for DNA or RNA
2.It is sensitive—can measure pg/mL; it is the recommended method for very diluted nucleic acid samples
3.It is accurate despite contamination being present in the sample, including nucleic acid contaminants |
| What are the disadvantages? | 1.It is not selective—uses software algorithms to distinguish mammalian DNA and RNA
2.It has limited sensitivity—detection limits are higher than fluorescence-based methods | 1.It takes longer—reagent and sample preparation are required
2.No purity information is provided |
