The effect of tissue decalcification on mRNA retention within bone for in-situ hybridization studies.
Tissue decalcification is a routine part of the preparation of bone tissue for histological studies. Although in-situ hybridization has been employed to localize mRNA of collagenous and non-collagenous bone related proteins in skeletal tissue, little is known regarding the effects of decalcifying agents on mRNA retention within tissue. In this study in-situ hybridization using an oligonucleotide probe (i.e. a poly d(T) probe) to detect total messenger RNA has been employed to investigate the effects of the decalcifying agents nitric acid, formic acid and EDTA on mRNA retention compared to undeacalcified tissue. The results show that formalin fixation and EDTA decalcification preserve substantial amounts of mRNA within the tissue. In particular, this study illustrates that it is possible to perform in-situ hybridization on formalin fixed decalcified paraffin embedded tissue.
Department of Rheumatology, University of Manchester, UK.
L
L
Walsh
A J
AJ
Freemont
J A
JA
Hoyland
D001842:Bone and Bones / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D003648:Decalcification Technique; D004492:Edetic Acid / Q000494:pharmacology; D005561:Formates / Q000494:pharmacology; D006801:Humans; D017403:In Situ Hybridization; D009566:Nitrates / Q000494:pharmacology; D017942:Nitric Acid; D012333:RNA, Messenger / Q000032:analysis* / Q000187:drug effects; D013048:Specimen Handling / Q000379:methods*; D016707:Tissue Fixation
D005561:Formates; D009566:Nitrates; D012333:RNA, Messenger; C030544:formic acid; D017942:Nitric Acid; D004492:Edetic Acid