From UV-Vis spectrophotometry and Fluorometery to qPCR, there are many different techniques by which RNA quantification can be carried out. Fluorometric RNA quantification provides sensitivity and specificity without the high capital costs or lengthy run-times of qPCR systems, making it the go-to choice in life science labs of all sizes.
What is RNA quantification and why is it important?
RNA quantification is the measurement of the concentration of RNA in a given sample. This process is fundamental in biochemical processing and is typically carried out as a preliminary step before further experimentation.
Downstream experiments typically rely on accurate RNA quantification in order to ensure valid and reproducible results. Without robust RNA quantification, variable sample input amounts are inevitable and this can have serious negative effects on the success and reproducibility of costly downstream applications. RNA quantification also acts as a safety net by confirming that extraction procedures have been successful and RNA is present in the sample before continuing with experimentation.
How do you calculate RNA concentration using fluorometric methods?
Fluorometric RNA quantification techniques rely on the use of fluorescent molecular dyes, which selectively bind to RNA molecules.1
Fluorescent molecular dyes can be engineered to exhibit various levels of specificity to a range of targets. While some bind indiscriminately to any single-stranded RNA molecule, others may be “site-specific”designed to bind to certain nucleotide sequences within RNA molecules to enable quantification of specific RNA sequences.2
Fluorometric RNA quantification assays are relatively straightforward. A dye is introduced to the RNA sample, at which point dye molecules (known as fluorophores) bind with target RNA molecules and are usually ready to analyze with minimal or no incubation time.
Once bound, these fluorophores are easily detected via optical means—this is usually carried out within an automated system known as a fluorometer. The fluorometer illuminates the sample with light of a specific wavelength capable of “exciting” the fluorophores. After excitation, the fluorophores will emit light at a characteristic frequency that the fluorometer detects. This emitted light is captured, and its intensity is measured in relative fluorescent units (RFUs). By comparing this intensity to that of a standard curve of known concentrations, RNA quantification can be rapidly and accurately carried out.
The ease with which fluorophores can be detected means that fluorometric RNA quantification techniques are around 1,000 times more sensitive than absorbance-based methods such as UV-Vis spectrophotometry. In addition to this, fluorometers typically represent much lower initial costs than qPCR systems, which, while sophisticated, are often prohibitively expensive and require greater levels of assay cost and hands-on time.
Fluorometric methods provide rapid RNA quantification that is accurate and comparatively low-cost. Because the method relies on the selective binding of fluorophores, fluorescence-based RNA quantification is highly selective and sensitive.
Fluorometric RNA quantification equipment from DeNovix
DeNovix is a leading provider of fluorometric RNA quantification systems, including the award-winning DS-11 Series and the QFX Fluorometer.
The DS-11 Series combines full spectrum UV-Vis analysis and fluorescence within a single compact unit to provide best-in-class sensitivity. In RNA quantification applications, the DS-11 Series detects as little as 0.60 ng/µL using nano volume UV-Vis or 250 pg/µL using fluorescence.
The QFX Fluorometer provides unmatched performance for all fluorometric applications where enhanced specificity and/or sensitivity are required. Easily carry out RNA quantification using four user-selectable fluorescent channels with a choice of common assays, or easily create and save custom programs for new fluorometric assays.
To find out more about DeNovix’s RNA quantification solutions, get in touch with a member of our team today.
