Analysis of gene expression in mineralized skeletal tissues by laser capture microdissection and RT-PCR.
The analysis of gene expression by growth plate chondrocytes in vivo has been hampered by the inherent difficulty in performing in situ hybridization on mineralized tissues. The combination of laser capture microdissection and reverse transcription-polymerase chain reaction (RT-PCR) allows analysis of gene expression by cells selectively removed from histologic sections by laser ablation. In order to apply this method to mineralized tissues, a decalcification process is required. The object of this study was to determine the optimal method for tissue decalcification prior to laser capture microdissection RT-PCR that will preserve integrity of the mRNA population. Acetone, 10% formalin, and methacarn were evaluated as fixatives, while Surgipath Decalicifier I, 10% ethylenediaminetetraacetic acid (EDTA), and 20% EDTA were evaluated as decalcifying reagents. Our results demonstrate that the optimal RNA quality was preserved by a decalcification protocol consisting of 20% EDTA for decalcification followed by fixation in methacarn, although this method is also associated with a reduction in RNA quantity.
Shao
Yvonne Y
YY
Orthopaedic Research Center, Department of Orthopaedic Surgery, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Wang
Lai
L
Hicks
David G
DG
Ballock
R Tracy
RT
D000818:Animals; D018775:Bone Demineralization Technique / Q000379:methods*; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D006132:Growth Plate / Q000166:cytology; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D019188:Organ Preservation Solutions; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation / Q000379:methods
D019188:Organ Preservation Solutions; D012333:RNA, Messenger