New approach in RNA quantification using arginine-affinity chromatography: potential application in eukaryotic and chemically synthesized RNA

The knowledge of RNA’s role in biological systems and the recent recognition of its potential use as a reliable biotherapeutic tool increase the demand for development and validation of analytical methods for accurate analysis of RNA. Affinity chromatography is a unique technique because of the versatility of applications reliant on the affinity ligand used. Recently, an arginine-based matrix has been effectively applied in the purification of RNA because of the specific recognition mechanism for RNA molecules. This interaction is suggested to be due to the length of arginine side chain and its ability to produce good hydrogen bonding geometries, which promote multi-contact with RNA backbone or RNA bases, based on RNA folding. Thus, this work presents the development and validation of an analytical method with ultraviolet detection for the quantification of RNA using affinity chromatography with arginine amino acid as immobilized ligand. The method was validated according to International and European legislation for bioanalytical methods. The results revealed that the proposed method is suitable for the reliable detection, separation, and quantification of RNA, showing that the method is precise and accurate for concentrations up to 200 ng/μL of RNA. Furthermore, the versatility of the methodology was demonstrated by its applicability in the quantification of RNA from different eukaryotic cells and in crude samples of chemically synthesized RNA. Therefore, the proposed method demonstrates a potential multipurpose applicability in molecular biology RNA-based analysis and RNA therapeutics.

Spectrophotometric flow-injection determination of nickel in biological materials

A flow injection method has been developed for the direct determination of free available Pb(II) and total Pb content in wine samples. The method is based on the chemical sorption of Pb(II), from pH 7 buffered solutions, on a packed polyurethane foam column, modified by addition of 2-(2-benzothiazolylazo)-p-cresol (BTAC). After this step, lead was directly eluted with a stream of 0.1 mol l(-1) HCl into an air C(2)H(2) flame in which lead was determined by atomic absorption spectrometry. Total lead was analyzed after sample digestion with nitric acid and hydrogen peroxide, being free available lead determined by direct sample on-line preconcentration and elution. The method provides a limit of detection (3sigma) of 1 mug l(-1) lead and relative standard deviation, which varies from 6 to 0.7% for lead concentration of 10 and 500 mug l(-1). Total content of lead in wine samples analyzed varied from 8 to 42 mug l(-1) being obtained free available values of Pb(II) under the limit of detection of the method. Recovery studies on natural wine samples, spicked with inorganic lead, evidenced the remaining capability of ligands, present in the wine, to avoid lead retention on the polyurethane foam loaded with BTAC.