The amount of genomic DNA is an important factor in achieving accurate assays. If the concentrations of DNA and RNA in the sample are insufficiently and incorrectly quantitated, there is a high tendency for the assay to result in experimental errors and impair the interpretation of genotyping results. Quantitating DNA and RNA samples before performing any assay can also help you save reagents and samples for further testing.
There are three several ways to Quantitate DNA and RNA. Listed below are the three most commonly used DNA and RNA quantitation methods in laboratories.
UV Spectroscopy
This is the most widely used method of quantitating DNA and RNA. UV spectroscopy uses an ultraviolet light to illuminate the chemicals that are present in the sample, almost similar to fluorescent spectroscopy. However, in this method chemicals absorb or reflect the light through electronic transition from the ground state (lowest energy level) to excited state (highest energy level). In fluorescence, light travels from the excited state to the ground state.
Things to remember when doing UV spectroscopy:
- Set up the spectrophotometer correctly when using reagents
- Always consult the instrument manuals to know the different ways to determine the background constant and use the right one.
- Observe extra care when using diluents as they may have differing properties that can affect the final results.
- Use a DNA sample with absorbance at 260nm
- Do a simultaneous reading at 280nm to determine the concentration of contaminating protein.
Absolute Quantitation
This method is used to quantify the amount of genomic DNA that is viable for amplification. It requires the creation of a standard curve using genomic DNA samples of known concentrations. The standard sample is pre-quantitated and validated using spectrophotometry or fluorometry. This method is used to compare the unknown samples to the known samples for quantification.
Fluorometric Analysis
This method quantitates DNA and RNA using different intercalating dyes. The dyes that are used have unique properties that bind specifically to double-stranded DNA. As they bind, they fluoresce brightly allowing the transition from double-stranded DNA to single-stranded DNA visible to scientists. The details below summarize the quantitation of DNA by fluorometric analysis using various intercalating dyes.
Hoechst dye #33258
- Has low affinity for RNA causing it to be more sensitive than spectrophotometric measurements
- Binds preferably to AT-rich DNA, which may affect the readings of DNA base composition
Ethidium bromide
- Not sensitive to the base composition of DNA and binds to RNA
- Capable of detecting DNA in nanogram amounts
- Ideal for very pure DNA with high GC content
Pico Green
- Capable of selectively detecting as little as 25pg/mL and up to 100ng/mL of dsDNA
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