Analysis of RNA stability and (-) strand content in viral infections using biotinylated RNA probes.
Non-radioactive biotinylated RNA probes specific for plus (+) and minus (-) sense RNAs of brome mosaic virus (BMV) were synthesized in vitro from a plasmid bearing a 200 base pair fragment complementary to the 3' terminus of each of the three genomic RNAs of the virus. Using virion RNAs isolated from BMV infected barley plants, the sensitivity of biotinylated RNAs as hybridization probes was compared with that of 32P-labeled probes in Northern hybridization assays. Although the sensitivity of biotinylated and 32P-labeled probes is similar (approximately 5 pg), the time required to detect the RNA bands was much less than for autoradiography; (-) sense RNAs could be detected in 30 min whereas 48 h or more were required by autoradiography. The value of biotinylated probes for following RNA stability was exemplified by the detection of supplied inocula in protoplasts 24 h post-inoculation. Quantitation of relative accumulation of progeny (+) and (-) sense RNAs by densitometry of the Northern blots probed with biotinylated RNAs paralleled that of radiolabeled probes. The application of these probes was extended to the detection of RNAs in barley protoplasts and BMV infected plant sap by dot hybridization. In these tests, viral RNAs were detected in as few as 250 protoplasts and sap dilutions up to 1:2000. The merits of these non-radioactive probes in monitoring the replication events by the detection and quantitation of mutant progeny RNAs of BMV are discussed.
Rao
A L
AL
Department of Biology, Texas A&M University, College Station 77843-3258.
Huntley
C C
CC
Marsh
L E
LE
Hall
T C
TC
D001710:Biotin; D015152:Blotting, Northern; D004279:DNA, Viral / Q000032:analysis; D003720:Densitometry; D009029:Mosaic Viruses / Q000235:genetics* / Q000254:growth & development; D009154:Mutation; D010944:Plants / Q000382:microbiology; D010957:Plasmids; D011523:Protoplasts / Q000648:ultrastructure; D012313:RNA / Q000032:analysis*; D015347:RNA Probes; D018075:RNA, Complementary; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity; D014779:Virus Replication / Q000235:genetics
D004279:DNA, Viral; D015347:RNA Probes; D018075:RNA, Complementary; D012367:RNA, Viral; D012313:RNA; D001710:Biotin