Analysis of gene expression in mineralized skeletal tissues by laser capture microdissection and RT-PCR.
Lab Invest
; 86(10): 1089-95, 2006 Oct.
Article
em En
| MEDLINE
| ID: mdl-16940962
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
/
Técnica de Desmineralização Óssea
/
Condrócitos
/
Perfilação da Expressão Gênica
/
Microdissecção
Tipo de estudo:
Evaluation_studies
Limite:
Animals
Idioma:
En
Revista:
Lab Invest
Ano de publicação:
2006
Tipo de documento:
Article
País de afiliação:
Estados Unidos