RESUMEN
The analysis of differential gene expression has become increasingly important in recent years. Typically, differentially expressed genes are identified in a primary screening procedure, yielding candidate genes whose differential expression has to be verified. We provide a highly sensitive, efficient and nonradioactive differential screening procedure to analyze numerous candidate genes in a single step. This comprises labeling of poly(A)+ RNA of the cell types analyzed with DIG Chem-Link and differential hybridization to the candidate genes fixed on dot blots. DIG Chem-Link allows, to our knowledge, for the first time efficient and direct nonradioactive labeling of RNA in vitro. Advantages of this method include extremely short exposure times and the feasibility to re-use the probes after prolonged storage. Using this procedure, we isolated several genes that are differentially expressed in maturing Langerhans cells.
Asunto(s)
ADN Complementario/genética , Genes/genética , Técnicas de Sonda Molecular , Sondas ARN/genética , Animales , ADN Complementario/química , Digoxigenina/química , Femenino , Regulación de la Expresión Génica , Biblioteca Genómica , Humanos , Células de Langerhans/citología , Células de Langerhans/metabolismo , Mediciones Luminiscentes , Masculino , Ratones , Ratones Endogámicos BALB C , Hibridación de Ácido Nucleico , Sondas ARN/química , Sensibilidad y EspecificidadRESUMEN
Northern blot analysis using radioactive probes is still the most common technique to determine the accumulation of transcripts in cells and tissues. The main disadvantages of this technique are the possible health hazard, inconvenience during handling, the high amount of RNA target necessary for detection, and difficulties with stripping and reprobing. In this paper, we propose an easily applicable protocol for Northern blot analysis of plant RNA with enhanced sensitivity using digoxigenin-, fluorescein-, or biotin-labeled in vitro transcripts derived from PCR products. Furthermore, we show the rehybridization of Northern blots with fluorescein as a second hapten, avoiding any stripping procedure.
Asunto(s)
Northern Blotting/métodos , Sondas ARN , ARN Mensajero/análisis , ARN de Planta/análisis , Biotina , Digoxigenina , Fluoresceína , Genes de Plantas/genética , Haptenos , Hordeum/genética , Hibridación de Ácido Nucleico , Hojas de la Planta , Reacción en Cadena de la Polimerasa , ARN sin Sentido , Trazadores Radiactivos , Sensibilidad y Especificidad , Moldes Genéticos , Transcripción GenéticaRESUMEN
Newcomers to the DIG System often inquire about the possibility of performing Northern blot hybridizations with nonradioactive techniques. With the following examples, we would like to share our protocol for performing highly sensitive Northern blots. This procedure strictly adheres to the standard procedures detailed in our manuals and pack inserts, and there are no special "tricks" required. As a target, we have used total human skeletal muscle RNA (Clontech). We selected two probes: beta-actin and a probe comprising the cDNA of the transcription factor CTF1, which expresses a low abundant mRNA. We used in vitro transcribed RNAs exclusively as probes because, during the development of the DIG System, we have found that RNA probes exhibit a 10-100-fold higher sensitivity with RNA targets than do DNA probes. They are also less prone to background problems caused by probe concentrations that are too high. For DNA probes, we recommend an optimal probe concentration of 25 ng/ml. Using a probe concentration that is even slightly too high (e.g., 1.5 fold) will dramatically increase the background. For RNA probes, we recommend an optimal probe concentration of 100 ng/ml, which will not lead to background problems. In the following examples, we describe all experimental details, starting from the gel run for the blot.
Asunto(s)
Northern Blotting/métodos , Digoxigenina/química , ARN Mensajero/metabolismo , Actinas/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Humanos , Músculo Esquelético/química , Factores de Transcripción NFI , Sondas de Ácido Nucleico/química , Sondas de Ácido Nucleico/genética , ARN Mensajero/genética , Reproducibilidad de los Resultados , Factores de Transcripción/genéticaRESUMEN
In recent years, the application of rare cutting restriction enzymes, the separation of resulting DNA fragments on pulsed-field gels and the subsequent Southern blot analysis using radioactively labeled probes have been standard laboratory methods to create long-range physical maps of complex genomes. The disadvantages of this technology are the hazardous handling risks when working with radioactivity and long exposure times. In this paper, we describe the use of nonradioactively labeled probes for single-copy sequence detection in a complex plant genome after pulsed-field electrophoretic separation of DNA fragments in the Mbp range. The approach avoids the use of radioactivity and also reduces the exposure time from one to seven days to approximately 2-3 h.