We are essentially doing EMSAs with RNA instead of DNA and it's been great for us to have the Odyssey CLx Imager because we are devoid of radiation
Dr. Andrea Kasinski
In the realm of small RNA therapeutics, Dr. Andrea Kasinski has played a part in ushering the first microRNA (miRNA) mimic into clinical trials. The Assistant Professor and her team at Purdue University are dissecting biological roles of miRNAs and channeling them into the pipeline of potential cancer therapeutics.
By employing miRNAs to target gene expression or by using them as drug targets (to prevent or enhance their function), Kasinski is developing novel approaches to treat non-small cell lung cancers (NSCLC). “There's been very minimal advancement in therapeutics that show good efficacy in NSCLC such that survival rates have not increased appreciably in the last few decades, as they have for some other cancers,” she says. Her group, in collaboration with a biopharmaceutical company, is testing a combinatorial miRNA formulation MRX34 in phase 1 clinical trials1.
The Kasinski lab is bustling with activity on all fronts: from discovery of miRNA function, and high-throughput screening for small molecule inhibitors, to designing delivery agents, and in vivo testing of potential miRNA therapeutics. The versatile Odyssey® CLx Imaging System is utilized in a number of fluorescence-based assays along this spectrum.
Multifaceted Platform for Assay Development
For validating small molecule inhibitors against miRNA-protein binding, Kasinski routinely performs Electrophoretic Mobility Shift Assays (EMSAs). “Once we get hits from our high-throughput screen, we cross-validate them as a secondary screen utilizing gel shift assays.” Their team labels the RNA of interest with infrared fluorophores and performs mobility shift assays to optimize buffer conditions, and calculate EC50 concentrations. “We are essentially doing EMSAs with RNA instead of DNA and it's been great for us to have the Odyssey CLx Imager because we are devoid of radiation,” she adds.
Further, Kasinski has devised an atypical assay by melding together EMSAs and Western blotting. “We transfer our EMSAs to a membrane and then we do a Western blot for protein detection on the Odyssey CLx Imager. These things have been done with DNA, but not that often with RNA.”
The system also provides convenient imaging for other applications. “I particularly like the ability to multiplex. We use it extensively for a lot of Western blots and also agarose gels with Syto® 60,” she says.
Safer, Fluorescence-Based Alternatives to Existing Protocols
We are using it as much as we can. Just the way the CLx System functions, and factoring in the cost of film and chemiluminescent reagents, the quality of the data and eliminating radioactivity was huge for me.
With the switch to the Odyssey CLx Imager, their lab has moved away from the hassles of handling hazardous chemicals involved with radioactive EMSA detection. “A lot of administrative work that goes along, like the time involved in getting approval, doing wipe tests every week, depositing off the radiation, or filling out a request for the ethidium bromide gel to be picked up has been eliminated. Not having those hazards in the lab is nice. It has been much easier to deal with some of the regulatory guidelines as well.” Kasinski has been able to successfully adapt their existing EMSA protocol to safer, fluorescence-based detection on the imager.
The lab's darkroom usage for Western blots and EMSAs has also trickled down to zero with the arrival of the Odyssey CLx System. “We don't use any darkroom whatsoever. We have gone from quite a bit to none at all.” she asserts.
The sensitivity and ease of use of the instrument, and the ability to quantify have been valuable to the lab. “We are using it as much as we can. Just the way the CLx System functions, and factoring in the cost of film and chemiluminescent reagents, the quality of the data and eliminating radioactivity was huge for me. It all culminated in a very good [buying] decision, and there are probably about six or seven labs at Purdue that are now coming over and using it.”
Researchers like Kasinski are designing novel therapeutic approaches to cancer with the help of the versatile Odyssey CLx Imaging platform.
1AL Kasinski, K Kelnar, C Stahlhut, E Orellana, J Zhao, E Shimer, S Dysart, X Chen, AG Bader and FJ Slack (2014). A combinatorial microRNA therapeutics approach to suppressing non-small cell lung cancer; Oncogene (2014), 1–9, 1 September 2014; doi:10.1038/onc.2014.282.
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