High-quality RNA preparation for transcript profiling of osteocytes from native human bone microdissections.
Osteocytes, the most abundant bone cell type with important roles in tissue maintenance and pathological aberrations such as observed in bone metastases, are enclosed within a highly compact, calcified extracellular matrix. This location complicates analysis in native bone, with the consequence that despite their importance their in vivo molecular physiology is only poorly understood. We have examined the possibility of isolating osteocyte RNA for transcript profiling from native, frozen bone instead of employing the formalin-fixed, paraffin-embedded, decalcified version routinely used in histology, providing chemically modified and highly disintegrated RNAs. Bone tissue was tape-assisted cryosectioned and fixed to glass slides by support of UV-flash-triggered adhesive polymerization followed by quick hematoxylin-eosin staining to generate a guidance image for microdissection. Using an UVa-nitrogen laser, matrix-enclosed osteocytes were either excised and catapulted into RNA preparation vials or freed of accompanying nonosteocyte cellular material. The influences of bone sectioning, staining, and osteocyte capturing procedures on the prepared osteocyte RNAs were analyzed and the method was optimized accordingly. The obtained osteocyte RNAs showed the expected expression pattern of marker genes (reverse transcriptase-polymerase chain reaction), and, following conversion into fluorescent-labeled cDNAs, led to transcript profiles (cDNAchips; 2600 genes) with scatter-graph geometries indicating suitability for high-confidence evaluation. With the approach described here we introduce a methodological way for the characterization of the in vivo molecular physiology of osteocytes by functional genomics.
Eisenberger
Sabine
S
Biochemische Zellphysiologie (A135), Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Hoppe
Godehard
G
Pyerin
Walter
W
Ackermann
Karin
K
D001842:Bone and Bones; D005404:Fixatives; D005629:Frozen Sections; D020869:Gene Expression Profiling; D006801:Humans; D042282:Microdissection; D020411:Oligonucleotide Array Sequence Analysis; D010011:Osteocytes; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling
D005404:Fixatives; D012313:RNA