Due to this, the description of the implementation and modifications made by this protocol used in the detection of a small RNA, as well as the explanation and discussion of the theoretical foundations of the modifications posed, are the objective of this work. Therefore, its use and implementation in studies of detection, characterization and functionality of new sR-NAs will be of great use. This new protocol increases the sensitivity and specificity in the detection of small RNAs by northern blot. Additionally, the use of radioisotopes ( 32P), the most common method for marking probes, with which all of the risks of radioactivity must be assumed, is replaced by the use of digoxigenin (DG). The immobilization of the RNA in the membrane, which is normally carried out with ultraviolet light (UV), is replaced with the use of EDC (1-Ethyl-3-(-3-dimethylaminopropyl) carbodiimide)). The modifications basically consist of replacing the DNA oligonucleotide probes with probes that contain locked nucleic acids (LNA). (2010), which integrates three separately developed methodologies that in turn modify three aspects of the process that determine the specificity and sensitivity of the northern blot. A new protocol for non-radioactive northern blot called LED (LNA-EDC-DIG) was reported by Kim et al. DNA probes marked with radioactive phosphorus ( 32P) are commonly used (Gurman S Pall & Hamilton, 2008 Varallyay et al. Then, it is incorporated into the membrane that contains the immobilized RNA, and finally, the hybridization with the complementary sequence is detected.ĭifferent protocols have been published for the northern blot methodology, which primarily vary in the design and marking of the probe. The probe with a complementary sequence to the RNA of interest is marked with radioactive and non-radioactive methods. The general northern blot process consists of total RNA extraction, separating it by size through electrophoresis, and transferring and immobilizing it on a solid surface (membrane). Therefore, it continues to be widely used. Northern blot is a direct methodology that permits the detection of the small RNAs, their precursory or intermediary sequences the evaluation of their properties of expression and establishment of their size ( Wang & Yang 2010). For the aforementioned limitations, the most used methods for the detection of sRNAs (RT-qPCR and northern blot) need to be constantly optimized to improve sensitivity, specificity of detection, and quantification. The methodologies used to detect sRNAs face great challenges imposed by their size (≤ 200 nucleotides) and a low level of expression of the small RNAs, which in some cases does not exceed the detection threshold for the techniques used until now. Therefore, there is still an interest and a need for the detection, characterization and validation of their function. The function of many of these sRNAs is still unknown. 2013 van Wolfswinkel & Ketting, 2010 Winter et al. 1993), the list of small non-coding RNAs has increased, re-porting the participation of some of them in biological processes, such as the cutting and splicing of exons, genetic translation, development, differentiation, cell death, metabolic control, antiviral defense, and transcriptional and post-transcriptional regulation of gene expression ( Gomes et al. Since the discovery of the first small RNA, lin-4 ( Lee et al. Small non-coding RNAs (sRNAs) are sequences involved in different constitutive as well as regulatory cell functions. Palabras clave: 1-ethyl-3-(-3-dimethylaminopropyl) carboniimide) Digoxigenina Northern blot no radioactivo Sondas LNA sRNAs El diseños de la sonda con la tecnología LNA, el marcaje de esta con Digoxigenina y por último la fijación del RNA a la membrana mediante 1-Ethyl-3-(-3-dimethyla-minopropyl) carboniimide (EDC) y finalmente se discuten los fundamentos teóricos de estos cambios. En este trabajo se describe la implementación de un nuevo protocolo para Northern blot no radioactivo, con modificaciones dirigidas a mejorar su sensibilidad y especificidad. A pesar de la baja sensibilidad del Northern blot, esta metodología continúa siendo de uso común en la detección de sRNAs porque permite detectar el RNA pequeño así como a sus precursores, razón por la cual se usa como una metodología complementaria en este tipo de investigaciones. El interés en la detección, identificación, y caracterización funcional de los pequeños RNAs no codificantes (sRNAs), ha generado la necesidad de optimizar las metodologías comúnmente usadas en su detección, la reacción en cadena de la polimerasa cuantitativa (RT-qPCR) y Northern blot, con el fin de que sean más sensibles y específicas.
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