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University of California San Francisco

5 ARTICLES PUBLISHED IN JoVE

Bioengineering

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Felix Moltzahn^1,2,3, Nathan Hunkapiller^1,2,4, Alain A. Mir⁵, Tal Imbar^1,2,6, Robert Blelloch^1,2,3,7

¹The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco , ²Center for Reproductive Sciences, University of California San Francisco , ³Department of Urology, University of California San Francisco , ⁴Department of Cell and Tissue Biology, University of California San Francisco , ⁵Fluidigm Corporation, Fluidigm Corporation , ⁶Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, ⁷UCSF - Helen Diller Family Comprehensive Cancer Center, University of California San Francisco

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

Medicine

Myo-mechanical Analysis of Isolated Skeletal Muscle

Peter E. Oishi^1,2, Sompob Cholsiripunlert³, Wenhui Gong², Anthony J. Baker⁴, Harold S. Bernstein^1,2,5

¹Cardiovascular Research Institute, University of California San Francisco, ²Department of Pediatrics, University of California San Francisco, ³Department of Biology, San Francisco State University, ⁴Department of Medicine, University of California San Francisco , ⁵Eli and Edythe Broad Center of Regeneration Medicine & Stem Cell Research, University of California San Francisco

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

Neuroscience

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Zhiqiang Dong¹, Mahendra Wagle¹, Su Guo¹

¹Department of Bioengineering and Therapeutic Sciences, Programs in Human Genetics and Biological Sciences , University of California San Francisco

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Bioengineering

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

David S. Booth¹, Agustin Avila-Sakar², Yifan Cheng²

¹Graduate Group in Biophysics, University of California San Francisco , ²Department of Biochemistry and Biophysics, University of California San Francisco

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Neuroscience

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Chao He¹, Jin I. Lee², Noelle L'Etoile³, Damien O'Halloran¹

¹Department of Biological Sciences and Institute for Neuroscience, George Washington University, ²Fred Hutchinson Cancer Research Center, ³Department of Cell and Tissue Biology, University of California San Francisco

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.