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Awwww, Ain’t That Sweet?

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There are many things that I love about summer…being out on the water,  puttering around in my garden, and local festivals top the list.  But the best, the absolute best, is the produce.  I could (and have!) spent hours at the farmer’s market, admiring the myriad of colors and varietals laid out under crisp white tents, fresh lemonade in hand and son in tow.  I love to cook, and this time of bounty makes me a bit crazy.  I want it all; the crisp asparagus of late spring, the early sweet peas, the summer squash.  The ultimate goal, however, for my husband and I, is to eat roughly our weight in fresh summer tomatoes.  I love every kind I can get my hands on, and I relish the flavor of each and everyone.

For 90% of the year in Wisconsin, we are left to deal with greenhouse, grocery store variety tomatoes whose slices are roughly equivalent to placing a piece of wet bread atop your sandwich.  They are mushy, mealy, and lack any semblance of flavor.  Every great while, you get one that is almost reminiscent of a real tomato, and it makes me long for the dog days of summer.

I know I’m not alone.  As a foodie, I try to eat in season with my brethren.  It’s November?  We eat winter squash!  Of course, that’s what in season, but every once in a while in the doldrums of winter, I’d like a proper BLT.  So what makes those summer tomatoes taste so much better?  Their environment?  Their origin?  Sure.  Grown locally outdoors , picked ripe, and eaten within a day or two of being plucked from the vine ensures the best flavor.  But, what’s inside those sweet tomatoes that makes them so good? Continue reading “Awwww, Ain’t That Sweet?”

Bisulfite Conversion and Next Gen Sequencing

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WebinarsIn my last entry, I gave a little summary of one of many techniques that are used to study DNA methylation patterns in a loci-specific fashion using the COBRA technique. This time, we’ll take a look at a high-throughput, genome-wide method for analyzing DNA methylation status using a next generation sequencing approache called bisulfite sequencing, or Bis-Seq. Continue reading “Bisulfite Conversion and Next Gen Sequencing”

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).

Continue reading “Mapping Protein-RNA Interactions in vivo Using the HITS-CLIP Method”

Fixed in the Past, Focus on the Future

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“I would do more with my samples, but it’s just not possible…I know there’s probably a wealth of information in there, but there is just no way to get it out…I’ve got blocks of tissue sitting in the lab, experiments I want to run, but no good way to get clean nucleic acids out.”

These are a few of the comments I heard when talking with scientists at the American Society of Human Genetics meeting last week in Montreal. They, and countless other researchers, are sitting on a treasure trove of information that may have been locked away a few months ago, a few years ago, or decades ago. I’m referring to formalin-fixed, paraffin-embedded (FFPE) tissue blocks. It is estimated that there are upwards of 400 million tissue blocks archived globally and scientists are clamoring to find ways to best utilize nucleic acids derived from these tissues in applications like qPCR, microarrays, and next generation sequencing.1  Continue reading “Fixed in the Past, Focus on the Future”