Aim—To develop methods of messenger RNA (mRNA) in situ hybridisation (ISH) for use with routinely processed bone marrow trephine biopsy specimens, decalcified using formic acid, and long term cultures in order to demonstrate sites of synthesis of mRNA encoding monocyte colony stimulating factor (M-CSF).
Methods—Biotinylated oligonucleotide probes, directed against target sequences within M-CSF mRNA, were hybridised with sections from bone marrow trephine biopsy specimens and detected using Streptavidin-biotin alkaline phosphatase complex formation. Validation of results included demonstration of total mRNA and unrelated mRNA species in adjacent sections, with appropriate negative controls. Minor technical modifications were required to perform ISH with long term bone marrow cultures.
Results—M-CSF mRNA was demonstrated successfully in trephine biopsy specimens and long term cultures. Biopsy specimens varied in their requirement for predigestion with proteinase K and in the strength of the final reaction product, presumably due to variation in fixation. M-CSF mRNA was present in myelocytes and promonocytes. No stromal production of M-CSF mRNA was detected in biopsy specimens. ISH using long term bone marrow cultures confirmed production of M-CSF mRNA by developing monocytes and macrophages. Weak M-CSF mRNA expression was also seen in stromal fibroblasts.
Conclusions—ISH can be performed successfully with formic acid decalcified bone marrow trephine biopsy specimens and long term cultures. The presence of M-CSF mRNA in myelomonocytic cells suggests that an autocrine mechanism contributes to monocyte differentiation. The absence of detectable M-CSF mRNA in biopsy stroma and its presence in stromal fibroblasts within bone marrow cultures probably reflects reduced sensitivity of ISH following tissue fixation and processing.
- Bone marrow
- in situ hybridisation
- growth factors
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