Products for the Study of Small, Non-Coding RNAs

Introduction

Beginning with the discovery of microRNAs in the early 1990s¹, small non-coding RNAs (sncRNAs) have proved to be a major class of regulatory molecules in both plant and animal genomes². They have been found to play crucial roles in development, genome organization, and in numerous disease processes, notably cancers^3-5. To date, several distinct classes of sncRNAs have been defined including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), 21U RNAs, endogenous silencing RNAs, repeat-associated RNAs (rasiRNAs), and trans-acting RNAs⁶. IDT offers a line of products uniquely suited for the study of these non-coding RNA species, including products for detection, direct cloning, sequencing, and knockdown/knockout.

References

1. Lee RC, Feinbaum RL, and Ambros V 1993 The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Celfl 75: 843-854.

2. Zhang B, Wang Q, and Pan X 2007 MicroRNAs and their regulatory roles in animals and plants. Journal of Cellular Physiology 210: 279-289.

3. Millar AA and Waterhouse PM 2005 Plant and animal microRNAs: similarities and differences. Functional and Integrative Genomics 5: 129-135.

4. Rana TM 2007 Illuminating the silence: understanding the structure and function of small RNAs. Nature Reviews Molecular Cell Biology 8: 23-36.

5. Massimo N, Ferracin M, Sabbioni S and Croce CM 2007 MicroRNAs in human cancer: from research to therapy. Journal of Cell Science 120: 1833-1840.

6. Carthew RW 2006 A new RNA dimension to genome control. Science 313: 305-306.

Detection

The most straightforward means of detecting small RNAs is by Northern Blot hybridization. Oligonucleotide probes are preferred in order to achieve suitable specificity for these short RNAs. However, traditional³²P end-labeled probes (made using polynucleotide kinase) often lack sufficient sensitivity to detect small RNAs, which are frequently present at very low levels. IDT’s StarFire^TM oligonucleotide labeling method permits generation of oligonucleotide probes with 10-fold increased sensitivity. StarFire^TM probes have been shown to detect rare RNA species such as splicing intermediates and miRNAs^1-3.

References

1. Behlke MA, Dames SA, McDonald WH, Gould KL, Devor EJ, and Walder JA 2000 Use of high specific activity StarFire™ oligonucleotide probes to visualize low-abundance pre-mRNA splicing intermediates in S. pombe. BioTechniques 29:892-897.

2. Sempere LF, Dubrovsky EB, Dubrovskaya VA, Berger EM, and Ambros V 2002 The expression of the let-7 small regulatory RNA is controlled by ecdysone during metamorphosis in Drosophila melanogaster. Developmental Biology 244:170-179.

3. Sempere LF, Sokol NS, Dubrosvky EB, Berger EM, and Ambros V 2003 Temporal regulation of microRNAs expression in Drosophila melanogaster mediated by hormonal signals and broad-complex gene activity. Developmental Biology 259:9-18.

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Small RNA Cloning

The most direct approach for discovering new small RNAs and new small RNA families is by cloning¹. This approach requires ligation of cloning linkers to the 5′ and 3′ ends of small RNA species that have been enriched from a total RNA purification. Greatly improved cloning efficiency is achieved using linkers that are pre-activated by adenylation². IDT now offers a line of products specifically designed to facilitate cloning small RNAs from any RNA source in any species. This product line includes the miRCat^TM small RNA cloning kit as well as the miRCat-33^TM modification for 5′ ligation-independent small RNA cloning³. Also available are the three ready-made linker oligonucleotides (Linker-1, Linker-2, and Linker-3), each containing different cloning sites for increased experimental flexibility, pre-activated at the 5′ end and 3′ end-blocked with a dideoxy-C (ddC). The 5′ M.R.S. Linker contains restriction endonuclease sites compatible with each of the 3′ linkers. Additional small RNA cloning tools offered include a 21-mer (miSPIKE™) and a 31-mer (piSPIKE™) internal RNA size and ligation control, and two sets of 454 Adaptor PCR primers designed to convert both miRCat^TM and miRCat-33^TM small RNA libraries into 454-compatible PCR libraries.

References

1. Berezikov E, Cuppen E, and Plasterk RHA 2006 Approaches to microRNA discovery. Nature Genetics Supplement 38: S2-S7.

2. Lau NC, Lim LP, Weinstein EG, and Bartel DP 2001 An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858-862.

3. Pak J and Fire A 2007 Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science 315: 241-244.

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miRNA Antisense Knockout

One proven means of investigating small RNA function is to transiently reduce expression levels by knockdown using antisense methods. In particular, use of 2′-O-methyl RNA oligos has met with recent success^1-2. IDT offers antisense oligos of a wide variety of chemical compositions, including 2′-O-methyl RNA and LNAs.

References

1. Meister, G., Landthaler, M., Dorsett, Y., and Tuschl, T. (2004) Sequence-specific inhibition of microRNAs and siRNA-induced RNA silencing. RNA, 10:544-550.

2. Hutvagner, G., Simard, M.J., Mello, C.C., and Zamore, P.D. (2004) Sequence-specific inhibition of small RNA function. PLoS Biology, 2:465.

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RNase Contamination

Nucleases are widely present in the laboratory environment and can interfere with many experiments. In particular, RNases can destroy valuable samples used in miRNA, siRNA, and other research that involves handling of RNA samples for the study of gene expression events. IDT offers a variety of products to help maintain a clean lab suitable for RNA research.

RNaseAlert^TM is a fluorescence-quenched RNA probe that allows for rapid visual or fluorometric detection of RNase contamination.

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RNase-free reagents are essential to RNA research. Making RNase-free reagents and testing them to ensure that they are RNase-free is a time-consuming process for small-scale labs. IDT offers inexpensive RNase-free reagents that undergo QC testing in large batches and can help you to keep your lab clean.

RNase enzymes are ubiquitous environmental contaminants and are employed in some molecular biology protocols and purified RNase stocks are usually kept and used in most labs, but unfortunately, RNase contamination is unavoidable. IDT offers a nuclease decontamination solution that can be applied to lab surfaces and will inactivate RNases and DNases.

Click Here to go to the Nuclease Decontamination solution Catalog Page