A microarray approach for comparative expression profiling of the discrete maturation zones of mouse growth plate cartilage.
In vertebrates, longitudinal bone growth is the consequence of a complex series of events that take place in a specialized structure, the growth plate cartilage. Within the growth plate chondrocytes undergo a sequential maturation program from resting cells to proliferative, pre-hypertrophic, and ultimately hypertrophic end-stage chondrocytes. This process of chondrocyte maturation is under the control of the temporally and spatially regulated expression of a myriad of signaling molecules, transmembrane receptors, transcription factors, and structural extracellular matrix (ECM) proteins. One approach to the comprehensive definition of the key components of such complex interrelated pathways is the use of microarray expression profiling to catalogue transcriptome changes during chondrocyte maturation in the individual developmental zones of the mouse growth plate cartilage. However, this has not been achieved because of the difficulty in obtaining sufficient quantities of the individual growth plate cartilage zones to all microarray analysis. In this study we describe the development of microdissection methods for the isolation of tissue from the proliferative, pre-hypertrophic, and proliferative zone from one single mouse femur, RNA extraction and linear amplification of the RNA to allow interrogation of NIA 15k microarrays to generate comparative expression profiles. Verification of a subset of differentially expressed genes by RT-PCR and by in situ hybridization confirmed the reliability of this approach.
Belluoccio
Daniele
D
Murdoch Childrens Research Institute and the Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville 3052, Victoria, Australia.
Bernardo
Bianca C
BC
Rowley
Lynn
L
Bateman
John F
JF
D000818:Animals; D002356:Cartilage / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D006132:Growth Plate / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D017403:In Situ Hybridization; D051379:Mice; D042282:Microdissection; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger