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A new approach for separating low‐molecular‐weight RNA molecules by staircase electrophoresis in non‐sequencing gels
Author(s) -
Velázquez Encarna,
Rivas Raúl,
Villar María del,
Valverde Angel,
Peix Alvaro,
Mateos Pedro F.,
Velázquez Enrique,
MartínezMolina Eustoquio
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500705
Subject(s) - rna , electrophoresis , resolution (logic) , ribosomal rna , chromatography , molecule , gel electrophoresis , biological system , chemistry , analytical chemistry (journal) , materials science , biology , biochemistry , computer science , organic chemistry , artificial intelligence , gene
Low‐molecular‐weight (LMW) RNA profiles, which include ribosomal and transfer RNA molecules with similar small sizes, are molecular signatures of microorganisms with a great potential in microbial identification. The greatest resolution of these profiles was achieved by staircase electrophoresis in sequencing gels. Nevertheless, this technique is difficult to use because it takes 7 h, the gels have large sizes and it is necessary to heat the system and to recycle the buffer to maintain the denaturing conditions and avoid smile effects. Most available sequencing slabs have no internal temperature control or homogenizing devices, which by contrast are present in some newly designed non‐sequencing slabs. Nevertheless, these slabs present two important problems for separating LMW RNA molecules, the size of gels is only 20 cm (instead of 40 cm) and the maximum voltage that can be reached is only 840 V (instead 2400 V). Staircase electrophoresis follows a model in which the external polarization is incrementally modified with a constant time step value. In the present work, we experimentally confirmed that by reducing the time step and increasing the total number of steps a suitable resolution is achieved. Under these conditions, despite the smaller size of the gels and the lower values of the electric field, the intensity reaches higher values than in sequencing gels and the LMW RNA profiles are correctly separated in 5 h. The resolution of these profiles obtained in non‐sequencing gels is similar to that obtained in sequencing ones facilitating the analysis of large populations of microorganisms in any laboratory.