Rapid and efficient generation of PCR-derived riboprobe templates for in situ hybridization histochemistry.
In situ hybridization histochemistry (ISH) using cRNA probes (riboprobes) has become a powerful technique for the examination of gene expression in tissue sections. The construction of plasmid templates for the synthesis of riboprobes with phage RNA polymerases is often a difficult and time-consuming step. We have therefore developed a rapid, efficient, and flexible method to generate totally artificial riboprobe templates by the polymerase chain reaction (PCR). We have made riboprobe templates using self-priming oligonucleotide primers spanning 146 BP of the 3' end of the human cytokeratin 1 (K1) gene coding region flanked by T7 and T3 promoters. These PCR-derived riboprobe templates were used to synthesize 35S-labeled anti-sense riboprobes as well as sense riboprobes as negative controls. The riboprobes were then applied in ISH to human skin sections made from routinely fixed and paraffin-embedded clinical biopsy material. Consistent with published results, we observed strong expression of K1 mRNA in the suprabasal cell layers of the epidermis but only weak to undetectable signals in the basal and cornified cell layers and in the dermis. With this experimental procedure we see no decrease in probe efficiency or quality compared to conventional methods. The use of PCR-derived riboprobe templates for ISH makes it possible to detect expression of any desired gene of known sequence rapidly and efficiently.
Sitzmann
J H
JH
Department of Dermatology, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
LeMotte
P K
PK
D001483:Base Sequence; D005796:Genes; D006651:Histocytochemistry; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D007633:Keratins / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D015347:RNA Probes / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis*; D012867:Skin / Q000737:chemistry
D015345:Oligonucleotide Probes; D015347:RNA Probes; D012333:RNA, Messenger; D007633:Keratins