= RNA melting baseline
{c}
An <RNA melting baseline> is the predicted <absorbance> of a specified folded or unfolded <RNA> population as a function of <temperature>, before mixing those populations in an <RNA optical melting> model. It differs from a solvent blank: a folded <RNA> sample still absorbs light, and that response can change with <temperature> even without the modeled transition.
For a <two-state RNA melting model> with unfolded fraction $f_U$, write
$$
A(T)=[1-f_U(T)]A_F(T)+f_U(T)A_U(T).
$$
The folded and unfolded <RNA melting baselines> might be modeled as $A_F=a_F+b_FT$ and $A_U=a_U+b_UT$. Their continuation into the transition region is <RNA baseline extrapolation>. Solving the optical mixture gives
$$
f_U=\frac{A-A_F}{A_U-A_F},\qquad A_U\ne A_F.
$$
For original stipulated readings $A=0.55$, $A_F=0.40$ and $A_U=0.70$, the inferred fraction is 0.50. Changing only the folded <RNA melting baseline> to 0.45 gives 0.40; changing only the unfolded <RNA melting baseline> to 0.80 gives 0.375. The same measured <absorbance> can therefore imply different populations under different baseline models.
Sensitivity is explicit. With $D=A_U-A_F$,
$$
\frac{\partial f_U}{\partial A}=\frac1D,\qquad
\frac{\partial f_U}{\partial A_F}=-\frac{1-f_U}{D},\qquad
\frac{\partial f_U}{\partial A_U}=-\frac{f_U}{D}.
$$
A small baseline separation amplifies <measurement uncertainty>. Correlated fitted baselines require covariance terms in <uncertainty propagation>, rather than independent error bars by assumption.<Evaluation of measurement data — Guide to the expression of uncertainty in measurement by JCGM>[§§5.1–5.2, sensitivities and correlated inputs.]{ref}
Optical-melting studies infer thermodynamics by fitting measured curves, rather than directly counting unfolded molecules.<Thermodynamic characterization of naturally occurring RNA tetraloops by Justin P. Sheehy and others>[Materials and Methods, individual-curve fitting.]{ref} Inspect the pre-transition and post-transition regions and test the chosen <RNA baseline extrapolation>. An inferred fraction outside $[0,1]$ signals noise or incompatibility with the assumed optical model; it is not a negative molecular population.
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