How do I perform absolute quantitation experiments using a standard curve?
Due to the short length of microRNAs, a standard curve for absolute quantitation can easily be generated using a synthetic microRNA target. In order for variations in PCR efficiency to be minimized, we recommend spiking the synthetic target into a complex RNA background (which does not contain the studied microRNA). Accurate determination of the concentration of the synthetic template is critical to the applicability of the standard curve. The absolute amount of microRNA in each experimental and control sample is interpolated from the standard curve, and is reported relative to a defined unit such as ng total RNA or copies per cell. Absolute quantitation does not exclude the use of an endogenous control for normalization. The abundance of the target microRNA(s) is normalized to the endogenous control (or a normalization factor calculated from multiple endogenous controls) in the same sample before absolute amounts are calculated using the standard curve.
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