Morgan OrsoliniDuke University | DU · Nicholas School of the Environment
Morgan Orsolini
Master of Science
About
7
Publications
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Introduction
Skills and Expertise
Publications
Publications (7)
Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte developmental competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is not only essential to employ superior methods of selecting high quality sperm, but also to be able to characterize which qu...
Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte quality and competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is essential to employ superior methods of selecting high quality sperm to maximize IVP outcomes. Clinical sperm selection techn...
As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of available semen selection techniques. This segment of our two-section review will focus on the selection of spermatozoa based on quality and sex for equine intracytoplasmi...
As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending...
Questions
Question (1)
Despite attempting several protocols that have been sufficient for extracting RNA in sperm from other species (Trizol, invitrogen) (Trizol/TCEP) and in equine (RNeasy, Qiagen), we have not been able to recover sufficient concentrations of RNA for RNA-seq. Samples were run through DGC for purification, diluted to cell numbers between 10 M and 40 M, and Omni mechanical homogenization was used for lysis. Rough estimates of concentration by Tapestation 4200 High Sensitivity RNA kit are 100-300 pg/uL (expected to be in the ng range), and no RIN values are able to be generated.
Has anyone had experienced troubleshooting with extraction of RNA from sperm and could offer some advice?
The attached document is our Tapestation report, samples being 20 M/mL, 40 M/mL, 60 M/mL, and 80 M/mL, with 0.5 mL used for extraction using the Trizol/TCEP prior to RNeasy kit. Sample was taken fresh, purified by DGC, and re-diluted into a concentration gradient.
