Northern blot

Páginas: 19 (4643 palabras) Publicado: 26 de abril de 2011
Uses of the Northern Blot

* Northern analysis is used for detecting and quantifying mRNA. Northwestern University defines northern blotting as the transfer of RNA molecules from a gel onto an absorbent sheet that is then immersed in a labeled probe that will hybridize to an RNA of interest. This reveals the RNA's presence. Northern blotting allows researchers to directly comparemRNA abundance on the absorbent sheets. Because of this, and its simplicity, this test remains the standard in RNA analysis.
* The thickness of the band, as well as the length the band of RNA has traveled during electrophoresis, indicates that the RNA of interest is present to some degree.
* The thickness and darkness of the band of RNA revealed by theprobe indicates the relative amount of RNA present in the sample. In addition, the length the band traveled during electrophoresis indicates the size, in kilobases (kb) of the RNA.
* Northern blotting allows researchers to determine gene expression patterns. This indicates a myriad of practical applications, allowing researchers to compare patterns of gene expression in cells oftissues, cells of patients undergoing treatment and cells of different developmental stages.
Northern blot analysis can also be used to detect cancerous pancreatic cells and tissues, as indicated by Streit, Michalski, Erkan, Kleeff and Friess. In one study they review, pancreatic cancers exhibited 3-fold, 15-fold and 10-fold increases in the mRNA of a certain receptor, indicating for the firsttime that this receptor was involved in the carcinogenesis of pancreatic cancer.

Análisis del Norte sigue siendo un método estándar para la detección y cuantificación de los niveles de mRNA a pesar de la aparición de técnicas de gran alcance, tales como RT-PCR, matriz de análisis genético y la protección de los ensayos de nucleasa. Northern analysis provides a direct relative comparison of messageabundance between samples on a single membrane. Análisis del Norte ofrece una relación de comparación directa de la abundancia de mensajes entre las muestras en una sola membrana. It is the preferred method for determining transcript size and for detecting alternatively spliced transcripts. Es el método preferido para determinar el tamaño de transcripción y para la detección de transcripcionesempalmados alternativamente.
The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (eg, integrity of the RNA sample and how efficiently it has transferred to the membrane). El procedimiento de borrar del Norte es sencilla y ofrece la oportunidad de evaluar los progresos realizados en varios puntos (por ejemplo, la integridad de lamuestra de ARN y la eficiencia con que ha transferido a la membrana). RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. muestras de ARN primero se separan por tamaño mediante electroforesis en un gel de agarosa en condiciones desnaturalizantes. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. El ARN setransfiere a una membrana, reticulado y hibridado con una sonda marcada. Northern hybridization is exceptionally versatile in that radiolabeled or nonisotopically labeled DNA, in vitro transcribed RNA and oligonucleotides can all be used as hybridization probes. hibridación del Norte es excepcionalmente versátil que nonisotopically o ADN marcado radiactivamente, en el ARN transcrito in vitro y losoligonucleótidos pueden ser utilizados como sondas de hibridación. Additionally, sequences with only partial homology (eg, cDNA from a different species or genomic DNA fragments that might contain an intron) may be used as probes. Además, las secuencias de homología con sólo parcial (por ejemplo, DNA de una especie diferente o fragmentos de ADN genómico que podría contener un intrón) puede...
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