Abstract
Application of a polyvinylalcohol-coated (PVA-coated) capillary in capillary gel electrophoresis (CGE) enables the selective separation of oligoribonucleotides and their modifications at high resolution. Quality assessment of shorter oligomers of small interfering RNA (siRNA) is of key importance for ribonucleic acid (RNA) technology which is increasingly being applied in medical applications. CGE is a technique of choice for calculation of chemically synthesized RNAs and their modifications which are frequently obtained as a mixture including shorter oligoribonucleotides. The use of CGE with a PVA-coated capillary to analyze siRNA mixtures presents an alternative to conventionally employed techniques. Here, we present study on identification of the length and purity of RNA mixture ingredients by using PVA-coated capillaries. Also, we demonstrate the use of PVA-coated capillaries to identify and separate phosphorylated siRNAs and secondary structures (e.g. siRNA duplexes).
Highlights
IntroductionThe most important and desirable characteristic for CGE capillaries and high pressure liquid chromatography (HPLC) columns alike is stability across the widest possible range of pH
Because there are no reports that unambiguously show the advantages of polyvinyl alcohol (PVA)-coated capillary for analysis of short synthetic RNA, we decided to show that short RNA is successfully separated by PVA-coated capillary
This article presents the results of our experimental studies showing the usefulness of the capillary gel electrophoresis technique using a PVA-coated capillary for qualitative and quantitative analysis of chemically synthesized oligoribonucleotides
Summary
The most important and desirable characteristic for CGE capillaries and HPLC columns alike is stability across the widest possible range of pH This feature is ensured by a capillary with polyvinyl alcohol as a permanent layer which is suitable for the analysis of the nucleic acids of biopolymers with a considerable number of sensitive moieties, because the PVA coating is stable from pH 2.5–9.530. Application of a PVA-coated capillary as hydrophilic nonionic coating proved to be useful in separating short-chain RNA from their phosphorylated analogs as well as oligomers of mixed sequences and defining their quality and purity. This technique enables the identification of such secondary structures as duplexes composed of complementary small interfering RNA (siRNA) strands from their primary structures
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