Estandarización de un protocolo para Northern blot no radioactivo usado en la detección de pequeños RNA en células Vero / Standardization of Northern blot non-radioactive protocol used in the detection of small RNAs in Vero line cells

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. 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. 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. 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-dimethylaminopropyl) carboniimide (EDC) y finalmente se discuten los fundamentos teóricos de estos cambios.

The interest in detection, identification and functional characterization of small non-coding RNAs (snRNAs), has generated the need to optimize the methodologies commonly used in its detection in specificity and sensitivity, The Quantitative reverse transcription PCR (RT-qPCR) and Northern blot. Even though the low sensitivity of Northern blot, this method continues to be commonly used in the sRNAs, because its capacity to detect the sRNA and its precursor, which is the reason why Northern blot is used as complementary method in this sort of Research. This work describes the implementation of an innovative non-radioactive Northern blot protocol, with modifications that improving the sensibility and specificity, with the discussion of the theoretical foundations of such modifications.

Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.

Diagnosis of Fragile X Syndrome and DMD by using DIG System / 대한산부인과학회잡지

The aim of this study was to develop a rapid and safe non-radioactive DIG DNA labeling and detection for Southern blot analysis for fragile X syndrome and Duchenne muscular dystrophy (DMD). Southern blot analysis is accurate test showing expression of the (CGG)n repeat and abnormal methylation pattern of CpG island in hagile X syndrome, and good confirmative secondary test in case of deletion in DMD. But in terms of test rapidity, these conventional radioactive Southern analysis may not be feasible for rapid screening of prenatal samples and at-risk populations to determine their status and to provide genetic counseling to their families. As an alternative radioactive Southern blotting, DIG DNA labeling and detection system does not require handling of radioactive material nor require learning any new technology. The complete procedure of labeling the DNA and hybridization to detection of the first visible signal can be compbsbed witbin 7 days. In addition, hybridization solutions containing labeled DNA can be reused several times after renewed denaturation.