Reduced formamide content and hybridization temperature results in increased non-radioactive mRNA in situ hybridization signals.
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.
Berndt
A
A
Institute of Pathology, Friedrich Schiller University Jena, Germany.
Kosmehl
H
H
Celeda
D
D
Katenkamp
D
D
D000199:Actins / Q000378:metabolism; D001483:Base Sequence; D004387:Dupuytren Contracture / Q000473:pathology; D005559:Formamides / Q000737:chemistry*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D015347:RNA Probes; D012333:RNA, Messenger / Q000737:chemistry*; D013696:Temperature
D000199:Actins; D005559:Formamides; D016376:Oligonucleotides, Antisense; D015347:RNA Probes; D012333:RNA, Messenger; C031066:formamide