Encarna Velázquez 1 Raúl Rivas 1 María del Villar 1 Angel Valverde 2 Alvaro Peix 2 Pedro F. Mateos 1 Enrique Velázquez 3 Eustoquio Martínez-Molina 1 1 Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca, Spain 2 Instituto de Recursos Naturales y Agrobiología, CSIC, Salamanca, Spain 3 Departamento de Física Aplicada, Universidad de Salamanca, Salamanca, Spain Received September 19, 2005 Revised November 6, 2005 Accepted November 6, 2005 Short Communication A new approach for separating low-molecular-weight RNA molecules by staircase electrophoresis in non-sequencing gels 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 incre- mentally modified with a constant time step value. In the present work, we experi- mentally 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 micro- organisms in any laboratory. Keywords: Electrophoresis / RNA / Microbiology / Taxonomy / Ecology DOI 10.1002/elps.200500705 The separation of nucleic acids (DNA and RNA) by elec- trophoresis is traditionally applied in microbiological and genetic approaches. Stable low-molecular-weight (LMW) RNA profiles were proposed as bacterial fingerprints by Höfle [1, 2]. They include 5S rRNA and tRNA in prokar- yotes [1, 2, 3, 6] and additionally the 5.8S rRNA in eukar- yotes [5, 6]. In 1997, we designed a new electrophoretic technique based on voltage steps, named staircase electrophoresis (SCE), for separating LMW RNA mole- cules [3], which has been recently applied to separate 16S–23S intergenic DNA fragments [4]. The separation of these molecules by SCE allowed the differentiation of fungi [5, 6], Gram-negative bacteria [6, 8–15] filamentous and non-filamentous Gram-positive bacteria [6, 16–18] and archaea [6]. Moreover, the LMW RNA profiles are suitable for the analysis of large microbial populations [8, 12–14], and allow the detection of new species and gen- era [9, 10, 15]. These results are in agreement with those obtained by ribosomal DNA sequences and DNA-DNA hybridization, and a wide study of prokaryotic and fungal species showed that LMW RNA are molecular signatures of microbial species [6]. Although the suitability of these profiles for bacterial classification and identification is currently accepted by microbiologists [7], the need for special conditions and equipment, which are not always available in most laboratories, has limited their use. Obtaining the LMW RNA profiles posed serious difficulties Correspondence: Dr. Encarna Velázquez, Departamento de Micro- biología y Genética, Laboratorio 209, Edificio Departamental, Univer- sidad de Salamanca, Avda. Campo Charro, s/n., 37007 Salamanca, Spain E-mail: evp@usal.es Fax: 134-23-224876 Abbreviations: LMW, low-molecular-weight; SCE, staircase electro- phoresis 1732 Electrophoresis 2006, 27, 1732–1738  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com