| Abstract |
Traditional thinking regarding gene regulation was shaken by the recent discovery of microRNAs, an abundant class of small, endogenous RNAs (19-25 nucleotides) that mediate post transcriptional regulation via components of the RNA-interference (RNAi) pathway (1), either by directing target transcript cleavage or by translational inhibition (2, 3, 4). Early microRNA discovery relied upon cloning and sequencing of small RNAs to find those whose primary configuration during transcription adopted a special hairpin structure; such a structure is an obligate precursor to the mature miRNA (5). Effective computational methods were later developed that are capable of discovering potential microRNAs based on different traits (hairpin structure, conservation among eukaryotes etc) (6, 12, 51). Hundreds of microRNAs have been discovered in complex eukaryotes, implying that this specific class mediates a vast network of unappreciated regulatory interactions, contributing to the complexity of gene regulation in higher organisms. Despite that hundreds of potential microRNAs have been predicted within the species, a small percentage seems to be indeed transcribed and a negligible number of them have known in vivo functions and biologically relevant target genes (2, 3, 17, 18). The need for substantiation of particular miRNΑs in the eukaryotes based on the output of the up-to-now computational approaches, led to the amendment of a technique that can lead to the imminent discovery of certain miRNAs with great sensitivity leaving behind time-consuming approaches (Northern technique) (7). This particular technique comprises a class of isothermal reactions that eventually lead to signal amplification of a particular miRNA, indirectly, in the form of 12-mers. The robustness, speed and sensitivity of the system may render the isothermal technique an indispensable tool for the discovery and the study of miRNAs in a broad spectrum of eukaryotic organisms. An important asset of this technique respectively, may as well be the quantification of the non-coding RNAs. An alternative approach for tracing small regulatory RNAs was developed in the lab. This approach led to the discovery of GFP siRNAs in cell extracts. siRNAs are chemically similar to miRNAs. They are implicated in gene regulation pathways, as well as protection from invasive genetic material (10, 11). Moreover, siRNAs participate epigenetic modifications of histones and DNA leading to transcriptional silencing (13, 14). This particular technique is based on the flexibility of theΤ7 polymerase to transcribe promoter-less templates, extending primers, eventually leading to the formation of DNA-RNA hybrids (8, 9). On the other hand, constructs that contain the GFP gene with binding site for miR399 (15, 16) in Arabidopsis, were cloned. Subsequent agroinfiltrations with the use of appropriate controls can provide information, not only for the presence of this particular RNA in the genome, but whether it has an active role in gene silencing through posttranscriptional interference.
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Ανάπτυξη τεχνικών για την άμεση ανίχνευση μη κωδικοποιών RNAs σε εκχυλίσματα ιστών
