This work was supported by the Institut National de la Sant&#233; et de la Recherche M&#233;dicale, the Centre National de la Recherche Scientifique, the Universit&#233; de Strasbourg, the H&#244;pital de Strasbourg, the Association pour la Recherche sur le Cancer, the Institut National du Cancer, the Agence Nationale de la Recherche and the R&#233;gion Alsace.
Pietro Laneve has been supported by the Fondation pour la Recherche M&#233;dicale. Currently, he is a recipient of a Istituto Italiano Tecnologia (IIT) fellowship. Publication costs are supported by the Neurex network (TriNeuron &#8211; Program Interreg IV Upper Rhine)

1. Sample Collection
<ol>
	<li>Detach semi-adherent Schneider&#8217;s 2 cells, (1-5 x 106 cells on average), by pipetting and harvest them by centrifugation (100 x g&#160;for 1 min). In case of in vitro cultured adherent cells, trypsinize them in standard conditions or directly collect them from plates into a convenient amount of RNA extraction reagent, by help of a cell scraper.</li>
	<li>For embryo collection, grow Drosophila strains in fly cages and let embryos accumulate on egg laying p...

Although our appreciation of the sophisticate intertwining through which RNA regulates neurogenesis is continuously increasing, the mechanistic implications of RNA-based circuitries affecting neural stem cell biology, neuronal differentiation/functional integration, development of neural pathologies and cancers remain unexplored. The maintenance of such networks through kingdoms makes the potential of genetic systems as Drosophila melanogaster instrumental to unravel unexplored cellular pathways, in which short ...

RNA biochemistry has experienced spectacular progresses in the past years1. Our comprehension of RNA potential in the control of gene expression has been burst by the identification of powerful noncoding ribo-regulators2, by the discovery of novel RNA-based regulatory mechanisms3,4 and by a deeper characterization of already known post-transcriptional events5. All together these studies have allowed RNA biology to dramatically make the scene, becoming a major research subject in the current scientific landscape. In particular, over recent years we are getting a sense of the pervasive impact of the &#8220;RNA world&#8221; on molecular neurobiology6, one of the most dynamic research domains in modern life science. In the last decade of the past century, the overall scientific scenario has been revolutionized by the discovery of the RNA interference7,8 and of the small regulatory RNAs9,10 with particular regard to microRNAs11, endogenously expressed small noncoding RNAs implicated in the control of nearly all cellular functions as pleiotropic and combinatorial regulators of gene expression.
Almost 10 years after miRNA initial discovery in Caenorhabditis elegans by Ambros and Ruvkun&#8217;s labs, renewed attention was turned to the field when high numbers of miRNAs were identified in Drosophila and in human cells as well12-15. Since then, thanks to versatile transgenic approaches, Drosophila melanogaster has stood out as a valuable biological context for delving into miRNA biosynthesis and activity. Drosophila miRNAs have revealed distinct functions in insect-specific or evolutionarily conserved processes, spanning from aging to metabolism, signaling pathways, behavior and, of course, neurogenesis. Along this direction, we recently unveiled a novel link16 in the intriguing correlation occurring between the master gene gcm/glide and the RNA pathway. The fly transcription factor Gcm/Glide17-19 constitutes a unique example of cell fate determinant, which dictates the glial vs. neuronal fate choice in multipotent fly neural precursors20. Twenty-year long research on this topic has clearly underlined the occurrence of multiple and overlapping inputs of gene expression regulation converging over gcm/glide21-28 to establish the threshold levels required for balancing the delicate ratio between neuronal and glial counterparts during neural development.
We discovered that regulation via Dmel-miR279 targeting represents a further control level contributing to post-transcriptional fine-tuning of gcm/glide expression16. Globally, these research lines have required specific methodological improvements: along years, several technologies originally developed to analyze traditional RNAs have been converted for quantifying small non coding RNAs, like as RNAse protection assays, cDNA arrays29-31, real-time PCR methods32-35 and sequencing36,37. On the other side, recalibration of technical approaches has fostered continuous progresses in the field.
Northern Blot assay (NB, or RNA gel blot assay) constitutes a representative instance: it is largely employed to profile RNA accumulation, since it ensures both expression level quantitation as well as size determination. However, the intrinsic poor sensitivity of the method is limiting when it is to be applied to low-abundant gene expression fine tuners, like short RNAs. A detrimental consequence is the requirement of large amounts of total RNA, which makes difficult its application to specific biological samples. For such reasons, specific NB variants for small RNAs detection have been developed38-40: we took advantage of an improved NB procedure41 (ENB, Enhanced Northern Blot), while elucidating the abovementioned interplay between Dmel-miR279 and gcm/glide.
This method relies on a chemical crosslinking step based on the activity of a Carbodiimide derivative [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC] to fix nucleic acid onto solid supports. Carbodiimide is a versatile cross linker known to catalyze the formation of amide bonds between activated carboxyl or phosphate groups and amine groups42. This property can be exploited to covalently couple small RNAs via their mono-phosphorylated 5&#8242;-hydroxyl group to amino groups at the surface of nylon membranes. The resulting attachment configuration increases the accessibility of the immobilized nucleic acid and, in turn, the efficiency of probe-target hybridization, which results in remarkable detection enhancement43.
The technique assumes particular relevance in Drosophila molecular studies, by which the occurrence of novel and distinctive classes of small noncoding RNAs is emerging44. Among these, rasiRNAs45,46 constitute a specific subtype of piwi-interacting RNAs (piRNAs47), involved in sequence-specific gene silencing. Operative details of this method are fully described and visualized hereinafter, relative to the analysis of the microRNAs Dmel-miR279 and Dmel-miR286 and, for the first time, of one rasiRNA, rasi4. We pushed to extremes this method, which allowed us revealing poorly expressed targets from minimal amounts of RNA (less than 1 &#956;g).

enhanced northern blot detection of small rna species in drosophila melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

By following the overall procedure described in the text and schematically represented in Figure 1, we assessed small RNAs expression from complex RNA samples of different origins. In the experiment presented in Figure 1, RNA was isolated from Drosophila Schneider&#8217;s 2 cells by Proteinase K extraction, checked for integrity (optional step: denaturing agarose gel fractionation) and loaded in double. One half of gel was blotted on a neutral membrane and chemically cross-linke...

Watch this Scientific Journal Video about Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster at JoVE.com

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

enhanced-northern-blot-detection-small-rna-species-drosophila

Research

JoVE Journal

Biology

23.7K vistas.  Institut de Génétique et de Biologie Moléculaire et Cellulaire.  El objetivo de esta publicación es visualizar y discutir los pasos operativos de un protocolo de transferencia Northern mejorada en el ARN extraído de Drosophila melanogaster embriones, células y tejidos. Este protocolo es particularmente útil para la detección eficiente de pequeñas especies de ARN. 