Researching Our Biospecimens

The tissue bank at the Windber Research Institute focuses on collecting and storing well annotated and high quality tissue and blood samples.  In order to demonstrate that the samples are of the highest quality, we do biospecimen research on samples in house to test different processing methods or storage conditions and determine which are best.  Pre-analytical variables that could affect downstream research processes that utilize DNA or RNA are studied in ongoing projects at our tissue bank.  High DNA/RNA concentration and good yield, integrity and purity are important in all experiments. 

            One such experiment is our biospecimen research project where we looked at the integrity and purity of RNA that was isolated from breast tissue collected from reductive mammoplasties.  Tissue specimens were processed by flash freezing or embedding in optimal cutting temperature (OCT) medium and either immediately stored at -80°C or kept at room temperature over varying time intervals.  RNA isolation was performed on all samples and the Nanodrop spectrophotometer was used to determine the concentration and purity of the RNA.  RNA integrity number was also determined using the Agilent Bioanaylzer.  Since laser capture microdissection is sometimes an integral part of tissue analysis, we also determined its effect on RNA integrity.  Quantitative Real-Time PCR was done for comparative measurement of the expression of four house keeping genes: B-actin, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin A and porphobilinogen deaminase.  Results of this study can be viewed on the additional poster.

A Cut above the Rest

Laser Microdissection is an intermediate step in the preparation of tissue specimens for research.  This service is provided by our tissue bank and gives researchers the ability to obtain macromolecules (DNA, RNA and protein) from specific cell types of a tissue specimen.  There are three Microdissection Microscopes currently in the market- Arcturus Pixcell, Leica Lasermicrodissection ASLMD and the Zeiss PALM.  We currently use the Leica ASLMD system.

Our primary focus in Windber is the study of breast tissue which is highly heterogeneous with numerous cell types within the primary tumor and the surrounding tissue.  Laser microdissection has allowed us to isolate pure samples from both invasive and in situ tumors (with and without involvement within invasive tumors), as well as the microenvironment surrounding the tumors that can then be used for downstream analyses.

We have used Laser Microdissected tissue samples for a variety of protocols requiring the preparation of homogenous isolates of DNA, RNA and protein.  Because the quality of RNA extracted from tissues can be affected by sample preparation and processing, we have fine-tuned our procedures for processing these samples.  Our protocols include careful cleaning of all equipment with RNAse inhibitors, use of special stains with lower water content, and rapidly performing the isolation process.  The time to process each sample is kept to less than 30 minutes from the time the specimen is sectioned through staining, microdissection and placement into the cell lysis buffer.  Below are images of a ductal carcinoma in situ (DCIS) from which RNA was isolated from the Microdissected cells.  The image labeled (A) is an Hematoxylin and Eosin Stain; (B) is a representative section in the same area stained with the RNA staining procedure and image (C) is the DCIS after Microdissection.

We have published details of our protocols in the chapter titled- “Laser Microdissection for Gene Expression Profiling” in the book “Laser Capture Microdissection Methods and Protocols Series: Methods in Molecular Biology”, Vol 755, Second Edition, Humana Press. 



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