CytoCite™ Fluorimetric Quantification vs. UV – Absorbance Measurements
Quantification of DNA or protein concentration in a sample is critical for the success of downstream applications such as next-generation sequencing, expression of proteins or qPCR. It can also prevent time consuming troubleshooting and reduce costly errors. For these reasons, scientists prefer a method of quantification that is high in sensitivity and accuracy.
Historically, DNA and protein quantification have been determined using spectrophotometry. This method uses spectrophotometers to measure concentration based on a molecule’s absorbance profile at specific wavelength: 260 nm for nucleic acids or 280 nm for proteins. Unfortunately, UV–absorbance suffers from poor selectivity and in certain applications can provide inaccurate readings because spectrophotometers are unable to discern between targets of interest and contaminants that absorb light at the same wavelength.
CytoCite™ fluorimetric quantification easily outperforms absorbance methods in sensitivity and accuracy. Two factors contribute largely to this, the first being instrumentation. The CytoCite™ Fluorometer has the capacity to measure samples with a high degree of linearity covering five orders of magnitude. This enable for more precise and accurate measurements of sample concentrations over a wider range.
Second, CytoCite™ fluorometer utilizes specially designed fluorescent dyes that are highly sensitive and selective to the target of interest. Because these dyes only fluorescence when bound to the target molecule – DNA or protein – fluorescence quantitation is not conflated with noise due to sample contaminants, free nucleotides or buffer elements. Another characteristic of CytoCite™ fluorescent dyes that contribute to assay sensitivity are their high extinction coefficients and fluorescence quantum yields. These properties enable dyes to generate significantly intense fluorescence signals that allow for the detection of molecules at three orders of magnitude lower than what is detectable by traditional absorbance.