Berndt A, Kosmehl H, Celeda D, Katenkamp D
Institute of Pathology, Friedrich Schiller University Jena, Germany.
Acta Histochem. 1996 Jan;98(1):79-87. doi: 10.1016/S0065-1281(96)80053-5.
To define conditions for highly sensitive non-radioactive mRNA in situ hybridization on cryostat sections the influence of decreased formamide content and hybridization temperature was studied. The examination was performed on fibromatosis nodules of palmar fibromatosis visualizing the beta actin mRNA of myofibroblasts. The results show that a decrease in formamide content and hybridization temperature is able to enhance the sensitivity of mRNA detection applicating digoxigenin labelled DNA oligodeoxynucleotide. The best hybridization signal could be obtained under formamide-free conditions. In conclusion, a simplified sensitive formamide-free mRNA in situ hybridization protocol using oligonucleotide probes on human tissue cryostat sections is presented. The negative formamide effect is seen as a result of the chemical interaction of formamide with nucleic acid strands. An omission of formamide is suggested if the target as well as the probe are single stranded.
为了确定在冷冻切片上进行高灵敏度非放射性mRNA原位杂交的条件，研究了甲酰胺含量降低和杂交温度的影响。对掌纤维瘤病的纤维瘤结节进行检测，观察成肌纤维细胞的β-肌动蛋白mRNA。结果表明，甲酰胺含量降低和杂交温度降低能够提高应用地高辛标记的DNA寡脱氧核苷酸检测mRNA的灵敏度。在无甲酰胺条件下可获得最佳杂交信号。总之，本文介绍了一种在人体组织冷冻切片上使用寡核苷酸探针的简化、灵敏的无甲酰胺mRNA原位杂交方案。甲酰胺的负面效应被认为是甲酰胺与核酸链化学相互作用的结果。如果靶标和探针都是单链的，建议省略甲酰胺。
