1. Compare and contrast the two main methodologies used
to calculate the concentration of nucleic acids. Namely,
spectrophotometry (Nanodrop) and fluorometry (Qubit). What are the
advantages and disadvantages of each? When would you use one over
the other?
2. RNA is labile and easily degradable. Thus, working
with RNA requires additional precautions and RNA is routinely kept
on ice to prevent degradation when preparing RNA extractions. Yet,
the human body temperature is 37°C and RNA is stable in our bodies.
Why is then RNA required to be kept on ice when handling it in the
lab?
thank you!:)
Answer: The Nanodrop technique utilizes the Ultra-violet absorbance and compares the absorbance ratio at 260 nm and 280 nm. Qubit fluorometry utilizes a specific dye that binds specifically to DNA and RNA.
Qubit fluorometry can be used to quantify both DNA and RNA from the same sample. While from Nanodrop spectrophotometry total nucleic acid (a combination of DNA and RNA) is quantified.
Qubit fluorometry estimates nucleic acid concentration accurately. Nanodrop spectrophotometry overestimates the concentration of nucleic acid and variations are often observed in the same sample when measured multiple times.
Contamination in the sample can provide an erroneous reading in the Nanodrop spectrophotometry. But, Qubit fluorometry shows less erroneous reading in the case of contaminations because of the use of dye specific to DNA and RNA only.
Quantification of nucleic acid using Nanodrop spectrophotometry is faster compared to Qubit fluorometry. For quick quantification, Nanodrop spectrophotometry is better.
Qubit fluorometry could not be used to quantify extremely low nucleic acid concentrations in the sample. In this case, Nanodrop spectrophotometry is a better option.
In Qubit fluorometry, it may so happen that all dyes are not bound to the nucleic acid. Since the measurement is actually done for the dye to estimate indirectly the nucleic acid, there is a chance of error sometimes. Nanodrop spectrophotometry measures the nucleic acid directly.
1. Compare and contrast the two main methodologies used to calculate the concentration of nucleic acids....
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