Bone Marrow Aspirate Paraffin Blocks as Reliable Source for Long-Term Protein Preservation.
Mehdi Nassiri, Sharon Ramos, Azorides Morales. Indiana University School of Medicine, Indianapolis; Univerity of Miami, FL
Background: Currently there is no economical method available to preserve bone marrow samples for molecular studies. We have studied feasibility of preserving biomolecule including proteins in bone marrow aspirate particle preparation using a novel molecular friendly tissue processing system.
Design: Bone marrow aspirates particle preparations were fixed in Molecular Fixative™ and processed using Tissue-Tek® Xpress™ (Sakura Finetek, Torrance, CA). Paraffin blocks were kept at room temperature for at least 18 months. Proteins were extracted from the freshly isolated samples, 50 micron thick sections of the paraffin blocks immediately after processing and after 18 months from the same block kept at room temperature. Commercial kits (Bio-Rad and Calbiochem) were used to isolate various subcellular compartments (nuclear, cytoplasmic organelles, cytoplasmic membrane) from the paraffin-embedded tissue. Isolated extracts were further separated on a fluid phase isoelectric focusing chamber (Bio-Rad Micro-Rotofor) and run on SELDI-TOF mass spectrometry on different protein-chip chemical surfaces. 2D gel electrophoresis with further spot excision and trypsin digestion was used to identify proteins using SEND ID chips. Immunohistochemistry was performed on paraffin-embedded tissue section for 20 major hematopoietic proteins.
Results: Protein profiling using mass spectrometry from paraffin embedded samples demonstrated similar pattern to fresh samples. Similar 2D gel pattern were obtained after 18 month interval from samples kept at room temperature. Immunohistochemistry was successful for all studied proteins.
Conclusions: It is feasible to use a novel formalin-free bone marrow particle preparation for preservation of high molecular weight macromolecules. Profiling using SELDI-TOF and specific protein identification were reproducible and similar after 18 month. This approach to preservation of bone marrow aspirate particles can serve as a cost-effective method for bio-repositories in clinical settings. Furthermore, preservation of bone marrow micro-architecture opens door for in situ molecular studies.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Monday, February 28, 2011 1:00 PM
Poster Session II # 253, Monday Afternoon