RNA

Working with RNA

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Set up a lab RNA Zone

Working with RNA can be a tricky thing…it falls apart easily, and RNases (enzymes that degrade RNA) are ubiquitous. Successfully isolating RNA and maintaining its integrity is critical, especially when sensitive downstream applications are used (e.g., RNA-Seq).

Good techniques for RNA handling are simple to employ but crucial for success. All RNA purification and handling should take place in an RNase-free, RNA-only zone of the lab. Segregating RNA work from protein and DNA purification and handling will help minimize the potential for RNase contamination and help keep your RNA intact. Only buffer and water stocks treated to be RNase-free should be kept in the RNA area of the lab, and gloves should be worn at all times to prevent accidental contamination. Tools and equipment such as pipets, tips, and centrifuges should be designated for use only in the RNA zone as well. The location of the RNA zone in the lab is also important. Keeping traffic to a minimum and moving the RNA zone away from doors, windows, and vents can also help minimize contamination.

Using an RNase inhibitor can also help safeguard your samples from RNase degradation. These inhibitors can bind to any RNases that may have been introduced into your sample and prevent them from cutting the RNA present.

Water and buffer stocks can be a source of RNase contamination. Several stocks from an RNase-free zone in an academic lab showed RNase activity. Recombinant RNasin® inhibitor protected all RNA samples from degradation.

Mapping Protein-RNA Interactions in vivo Using the HITS-CLIP Method

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RNA recognition domain from RNA Binding Protein 19 (source: protein database www.pdb.org)

Aberrant RNA binding protein (RBP) function has been implicated in a host of human diseases from various cancers, neurological disorders, and conditions related to muscular atrophy (1). Understanding RBP function requires not only a working knowledge of the protein proper, but accurate methods to identify RNA binding partners in vivo.  Identification of RNA binding partners has historically been difficult, especially for RNA targets involved in nervous system disorders. Methods for finding targets have involved in vitro RNA selection or co-immunoprecipitation followed by gene chip analysis (2,3). These approaches came with some inhert limitations. The signal to noise ratio is low and the ability to differentiate between direct and indirect interactions is limited. Additionally, since the RNA-protein interactions are so complex, any of the in vitro methods may not be wholly predictive of true intracellular interactions.

In 2003, researchers at the Laboratory of Molecular Neuro-Oncology at Rockefeller University developed a method to purify protein-RNA complexes from mouse brain tissue that utilized ultraviolet cross-linking of RNA to their protein binding partners and immunoprecipitation of the cross-linked product (4). Further development of the technology has resulted in a streamlined protocol to perform high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP; 5).

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