• Quantitating RNA through UV Spectroscopy

    Quantitating RNA is an important step before an RNA analysis. The amount of RNA can vary depending on the methods use in RNA sequencing. Knowing how much concentration of RNA is present in the sample will help scientists measure the rate of contamination and achieve accurate results in different assays.

    One of the most common ways to quantitate RNA is through UV spectroscopy. Also known as the traditional method, UV spectroscopy uses DNA sample with an absorbance ratio of 260nm. The process involves exposing the sample under the ultra-violet light. The absorption ratio of 260nm gives way for the light to pass through the sample, allowing the chemicals that are present in the sample to glow as they absorb and reflect the light.

    The concept of UV spectroscopy is based on the Beer-Lambert law, where two physicists, August Beer and Johann Lambert, explained that the light absorption is directly proportional to the properties of the material through which light is traveling. The RNA absorbs light because it has a high degree of conjugation, a system of interconnected p-orbital (the wave-like behavior of RNA) that makes it very easy to react with electromagnetic radiation.

    UV spectroscopy is the easiest way to quantitate RNA; however it also has its own drawbacks.

    • Due to addition of absorbance, the method has limited use in analyzing mixtures
    • Requires special equipments
    • Not selective for compounds if they absorb at the same wavelength
    • Does not discriminate between RNA and DNA

    Listed below are the advantages of UV spectroscopy.

    • Provides very high accuracy
    • Useful for a wide variety of chemicals
    • Non destructive to RNA
    • Can be used for quantitative and qualitative assay for pure substances

    Listed below are experts’ advices to minimize the drawbacks of quantitating RNA through UV spectroscopy.

    • Remove contaminating DNA prior to the assay by treating RNA samples with RNase-free DNase.
    • Remove residual proteins and phenol, which may alter the absorbance readings.
    • Clean the cuvettes before placing the samples.
    • Use buffered solutions with slightly alkaline pH to assure accurate reproducible readings.
    • Make sure the RNA dilution is within linear range. Solutions that are outside the range cannot be measured.
    • Treat RNA samples with RNase-free DNase to remove contaminating DNA.

    Categories: DNA & Genetics

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