The Laser Capture Microdissection (LCM) Resource/Facility at the University of Maryland serves as a resource to the NICHD SCCPIR network by providing the isolation of specific cells of interest from heterogenous tissues of the reproductive system, e.g. ovary, uterus, testis, brain, pituitary. The qualitative and/or quantitative analysis of RNA or DNA (e.g. RT-PCR, microarray, cDNA libraries) for factors of interest can then be performed on extracts of LCM-isolated cells. As methods for LCM advance, it is anticipated that protein quantification procedures will also be applied to isolated cell fractions.
SCCPIR PI: Eugene D. Albrecht, Ph.D.
Center for Studies in Reproduction
University of Maryland School of Medicine, Bressler Research Laboratories 11-019
655 West Baltimore Street, Baltimore, Maryland 21201
tel 410-706-3391, fax 410-706-5747, e-mail: ealbrech@umaryland.edu
LCM Coordinator: Donna Suresch, M.S.
SCCPIR Cell ICC: ISH Core
University of Maryland School of Medicine, Bressler Research Laboratories 11-016
655 West Baltimore Street, Baltimore, Maryland 21201
tel 410-706-4155, fax 410-706-5747, e-mail: dsuresch@som.umaryland.edu
Immediately freeze tissue sections (approximately 3-5 mm3) on cryomolds in a slurry of dry ice: isopentane and store in liquid nitrogen. Complete the attached LCM form specifying which cells are to be captured, the particular RNA and/or DNA analyses to ultimately be performed (in user’s laboratory), and the extraction buffer to be used. Carefully pack tissue samples in styrofoam container with ample dry ice and then within secure cardboard box and express ship overnight to LCM Coordinator Donna Suresch (address above). Note: If an unusual reagent is to be used for cell extraction (e.g. other than Qiagen RNeasy for RNA or Qiagen DNeasy for DNA), this reagent should be shipped simultaneously with the tissue samples.
The University of Maryland LCM Facility will: (1) section tissues in a cryostat at approximately 8 mm thickness; (2) lightly stain tissue sections with hematoxylin and eosin; (3) isolate cells of interest via LCM using the Arcturus Pixcell II Microdissection System; (4) extract RNA and/or DNA; and (5) ship extracted RNA and/or DNA on dry ice overnight to user. The volume or area of cells captured will be recorded to allow quantification.
In many cases the identification of cells to be captured, e.g. ovarian granulosa cells, uterine glandular epithelial cells, will be straight forward and not require overly extensive communication between the user and LCM Facility technician. In other instances, the isolation of specific cells of interest will be facilitated by use of Microsoft Net-Meeting software program. At the time of cell capture by LCM at a mutually (user and LCM Facility) arranged time the user and University of Maryland SCCPIR LCM Facility will establish a Net Meeting. In almost real time, the cells of interest can be simultaneously imaged on the computer monitors of the user and LCM Facility technician for purposes of directing cell capture. Contact Dr. Andrea Niklaus for technical assistance with cell capture and virtual network computing.
Requests for LCM cell isolation by SCCPIR users will be completed on a first-come/first-serve basis.
Note: NICHD has provided funds for only 50% time of the LCM technician during the first year of operation.
The University of Maryland SCCPIR LCM Resource received funds from the NICHD for technical personnel, LCM caps, and internet access. However, costs for cell extraction and staining reagents, slides, and return shipping of extracted samples will be invoiced and must be paid by the user via purchase order or check made payable to the University of Maryland.