sepharose and tetramethylrhodamine-isothiocyanate

A new method has been developed to detect RNA-DNA hybrids in situ by fluorescence microscopy. This overcomes some of the disadvantages of autoradiographical detection of in situ hybridization, notably the low resolution and long exposure times needed. A procedure to label RNA at its 3'-terminus with a fluorochrome molecule has been developed. The optimal conditions for the cytochemical hybridisation reaction of this fluorochrome-labeled RNA were investigated using a model consisting of Sepharose beads to which nucleic acids has been bound. With RNA labeled both with 3H and rhodamine the hybridization reaction could be studied both biochemically by scintillation counting and cytochemically by microfluorometry. The fluorochrome-RNA bond was found to be unstable at higher temperatures. Therefore, the hybridization reaction had to be performed at room temperature in formamide-containing buffers. With fluorochrome-labeled complementary RNA kinetoplast DNA in Crithidia luciliae, adenovirus-5 DNA in infected KB cells and 5S rRNA, tRNA and cloned histone genes in polytene chromosomes of Drosophila could be localised. An immunocytochemical amplification method was developed that increased the sensitivity of the direct method. Several recently developed hybridocytochemical methods based on a combination of a hybridisation reaction in the first layer followed by immunocytochemical second layers, are described. The prospects of the application of the hybridocytochemical techniques in biomedical research, such as gene localisation and virus diagnosis, are discussed.

Topics: Autoradiography; DNA; Fluorescein-5-isothiocyanate; Fluoresceins; Histocytochemistry; Microscopy, Fluorescence; Models, Chemical; Nucleic Acid Hybridization; Nucleic Acids; Rhodamines; RNA; Sepharose; Thiocyanates

A new procedure to label RNA at the 3'-terminus with a fluorochrome molecule is described. The thiosemicarbazides derived from tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC) were prepared by reacting these compounds with hydrazine in dimethylsulfoxide (DMSO):pyridine, 99:1 (v/v). They coupled efficiently to the aldehydes generated by periodate oxidation of RNAs. We determined, using Sepharose to which different nucleic acids and proteins had been bound, that the label added no specific binding properties to the RNA, and did not interfere with duplex formation of labeled poly(U) and poly(A). The stability of the fluorochrome-RNA bond under conditions generally used for hybridization was investigated. The bond was found to be unstable at 66 degrees C in 3 x SSC, 0.1% SDS (50% loss within 45 min) but stable for at least 40 hr at 23 degrees C in 70% formamide/3 x SSC. The hybridization characteristics of complementary RNA, both fluorochrome- and 3H-labeled, were investigated using DNA-Sepharose beads as a cytochemical model. Hybridization was measured by scintillation counting of microliter quantities of beads and quantitative fluorescence microscopy of individual Sepharose beads. No influence of the label on the specificity and stability of the hybrids was found. Maximum specific fluorescence was found after hybridization at 23 degrees C in 70% formamide/3 x SSC. These results made possible the successful use of fluorochrome-labeled RNA to perform cytochemical hybridization followed by detection of the hybrids with fluorescence microscopy. This will be described in an accompanying article.

Topics: Coloring Agents; DNA; Fluorescein-5-isothiocyanate; Fluoresceins; Histological Techniques; Microscopy, Fluorescence; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Poly A-U; Polyribonucleotides; Rhodamines; RNA; Sepharose; Thiocyanates