Rapid in situ hybridization technique for the detection of ribonucleic acids in tissues using radiolabelled and fluorescein-labelled riboprobes.
In situ hybridization (ISH) is a useful diagnostic and research tool, but is also time consuming. This study was conducted to determine if a rate enhancement hybridization (REH) buffer, developed for membrane hybridization, could be used to decrease hybridization time for ISH. Tissue from swine with an enteric disease produced by a swine coronavirus, transmissible gastroenteritis virus (TGEV), was used as a model to standardize hybridization conditions for a rapid ISH technique. Small intestinal sections from pigs experimentally and naturally infected with TGEV were hybridized for various times at 52 degrees C and 70 degrees C with a radiolabelled or a fluorescein-labelled RNA probe in a standard hybridization or a REH buffer. Viral RNA was detected in intestines from as early as 30 min of hybridization by using both buffers with the radiolabelled probe; however, the signal was stronger with the REH buffer. With the fluorescein-labelled probe, viral RNA was detected in virus-infected cells of the intestines after 30 min of hybridization by using the REH buffer. Signal intensity was greater with the REH buffer than with the standard hybridization buffer when compared at each hybridization time and hybridization temperature using both radiolabelled and fluorescein-labelled probes. With the REH buffer, hybridization signal intensity was greater at 70 degrees C than at 52 degrees C for both probes. The best results were obtained when small intestinal sections were hybridized at 70 degrees C for 2 h using a radiolabelled or a fluorescein-labelled probe diluted in the REH buffer. The fluorescein-labelled RNA probe with REH buffer resulted in a minimal non-specific signal when compared with the radiolabelled probe. These studies demonstrated that the REH buffer can be used to decrease the time of ISH for the detection of viral RNA. This rapid ISH technique should have broad applications in the utilization of probe technology in diagnostics and research for the detection of target ribonucleic acids in situ
Sirinarumitr
T
T
Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
Paul
P S
PS
Halbur
P G
PG
Kluge
J P
JP
D000818:Animals; D002021:Buffers; D019793:Fluorescein / Q000737:chemistry*; D005759:Gastroenteritis / Q000821:virology; D017403:In Situ Hybridization / Q000379:methods*; D007422:Intestines / Q000473:pathology / Q000821:virology; D012313:RNA / Q000737:chemistry; D012265:Ribonucleotides / Q000032:analysis*; D013552:Swine; D013696:Temperature; D005760:Transmissible gastroenteritis virus / Q000235:genetics / Q000302:isolation & purification
D002021:Buffers; D012265:Ribonucleotides; D012313:RNA; D019793:Fluorescein