PubMed · 11124051
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I M Verma. 2000. Gene therapy: the need for basic science.. https://doi.org/10.1006/mthe.2000.0220
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STALARD (selective target amplification for low-abundance RNA detection) enables isoform-level quantification of low-abundance RNAs using conventional laboratory equipment. Here, we describe steps for RNA isolation, primer design, reverse transcription, selective target amplification, and downstream analysis. The protocol couples selective pre-amplification with a quantitative reverse-transcription PCR (RT-qPCR) readout and optional nanopore sequencing. Using 1 μg input RNA and 12 pre-amplification cycles, STALARD reduces Cq values by approximately 10-12 cycles, bringing the target into a reliably quantifiable range. For complete details on the use and execution of this protocol, please refer to Jeong et al.1.
The analytic solution to the nonlinear Poisson-Boltzmann equation describing the ion distributions surrounding a nucleic acid or other cylindrical polyions as a function of polyion structural quantities and salt concentration ([salt]) has been sought for more than 80 years to predict the effect of these quantities on the thermodynamics of polyion processes. Here we report an accurate asymptotic solution of the cylindrical nonlinear Poisson-Boltzmann equation at low to moderate concentration of a symmetrical electrolyte (< or = 0.1 M 1:1 salt). The approximate solution for the potential is derived as an asymptotic series in the small parameter var epsilon(-1), where var epsilon identical with kappa(-1)/a, the ratio of the Debye length (kappa(-1)) to the polyion radius (a). From the potential at the polyion surface, we obtain the coulombic contribution to the salt-polyelectrolyte preferential interaction (Donnan) coefficient (Gamma(u)coul) per polyion charge at any reduced axial charge density xi. Gamma(u)coul is the sum of the previously recognized low-salt limiting value and a salt-dependent contribution, analytically derived here in the range of low-salt concentrations. As an example of the application of this solution, we obtain an analytic expression for the derivative of the midpoint temperature of a nucleic acid conformational transition with respect to the logarithm of salt concentration (dT(m)/d ln[salt]) in terms of [salt] and nucleic acid structural quantities. This expression explains the experimental observation that this derivative is relatively independent of salt concentration but deviates significantly from its low-salt limiting value in the range 0.01-0.1 M.
Iron and phosphorus are essential minerals for both humans and plants. Advances in our understanding of the molecular mechanisms involved in the mobilization, transport and storage of these minerals now allow us to engineer plants to improve the yield and mineral nutrition of crops. Strategies range from increasing the expression of endogenous genes, such as that encoding the iron storage protein ferritin, to expressing a phytase gene from the fungus Aspergillus in Arabidopsis, thereby allowing the plants to obtain a previously unusable pool of phosphorus.