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In molecular biology, Nucleic acid (DNA and RNA) quantification is a critical step in determining the concentration of DNA or RNA in a sample prior to downstream experiments. The success of transfection, molecular cloning, next generation sequencing, and nucleic acid amplification analysis depends on accurate determination of nucleic acid concentration and yield. Inaccurate quantification can increase the variability of downstream assays and affect the quality of results. Quantitative methods for DNA and RNA include photometry (UV-Vis), fluorometry, agarose gel electrophoresis, and qPCR. Among them, UV-Vis measurement and fluorescence measurement are commonly used to quantify nucleic acids.

UV-Vis measurement has been a routine method for DNA and RNA quantification and can be used to estimate the concentration of DNA or RNA in purified samples. This method is simple and does not require further sample processing, other than DNA/RNA extraction, and does not require reactions with other substances. However, the UV-Vis method is difficult to detect nucleic acids with low concentration in samples and is susceptible to contaminants.

Fluorometry is one of the most sensitive methods for nucleic acid quantification. This method can use specific fluorescent dyes to bind specifically to specific types of nucleic acids, such as dsDNA, ssDNA, RNA, or small RNA. The dyes themselves have low intrinsic fluorescence and emit light when they are bound to biomolecules. Each nucleic acid dye has binding and spectral properties. The specificity of the fluorometric method makes it 10 to 1000 times more sensitive than the photometric method for quantifying nucleic acids. This feature allows the fluorometric method to measure the concentration of nucleic acids even in highly diluted samples.

Agarose gel electrophoresis is a quantitation method that can separate DNA and RNA from other impurities. In agarose gel electrophoresis, negatively charged nucleic acids migrate toward the anode, separating DNA and RNA fragments based on size and shape. Fluorescent dyes such as ethidium bromide can be added to the gel or sample, followed by electrophoresis. The bands in the gel can be visualized by a UV transilluminator or gel imaging system. The amount of nucleic acid in a sample can be estimated by comparing the intensity of a sample band with standards of known amounts. Because the method is measured by visual inspection or using image analysis software, it has low sensitivity, low accuracy, and takes a long time.

Real-time PCR or quantitative PCR (qPCR) quantifies the DNA using fluorescent probes that bind the target DNA in the annealing stage of PCR or fluorescent dyes that bind double-stranded DNA. A specialized thermal cycler equipped with a fluorescence detection module is used to monitor the fluorescence signal. The measured fluorescence is proportional to the total amount of DNA. A standard curve is used to determine the amount of target DNA in the sample. With digital PCR, DNA quantification can be performed without a standard curve.

Amerigo Scientific offers quantification kits based on photometry, fluorometry, or agarose gel electrophoresis for determination of DNA, RNA, or specific nucleic acid concentrations. Our products are designed to accurately determine the concentration of target nucleic acids in a short time for optimal performance of downstream assays.