Use of acridine orange staining for the detection of rotavirus RNA in polyacrylamide gels.
Acridine orange is a metachromatic intercalator used extensively in histochemistry to differentiate double- from single-stranded (ds, ss) nucleic acid by the emission of green and red fluorescence, respectively, under ultraviolet light. In the present study we standardised a protocol in order to use acridine orange to detect rotavirus ds RNA in polyacrylamide gels and compared it to silver and ethidium bromide staining. We demonstrated that the simplest and best condition was attained when gels containing rotavirus ds RNA bands, stained in green, were treated with 4.3 microM acridine orange after electrophoresis and destained with distilled water pH 6 at 37 degrees C. Under this protocol, rotavirus RNA concentration was calculated and the mean minimum amounts of nucleic acid detected by acridine orange, ethidium bromide, and silver staining were 26.0 +/- 4.29, 15.6 +/- 1.48 and 1.06 +/- 0.11 ng, respectively. The comparison of acridine orange sensitivity with ethidium bromide and silver staining, for 25 field strains of rotavirus and one cell-adapted strain (SA11), demonstrated concurrent results in 80% of the specimens. Red colour emission resulting from the interaction of acridine orange with ss nucleic acid was also shown by testing denatured 0.5 kb HindIII digest of lambda phage DNA. Furthermore, it was demonstrated that rotavirus ds RNA could be used for reverse transcription activity, followed by PCR amplification, after acridine orange staining. In conclusion, although acridine orange is less sensitive than ethidium bromide and silver staining, its practicality, low cost, metachromatic properties, and its non-interference on RT-PCR should be considered. It is suggested the use of acridine orange as an appropriate stain for various purposes in virology, as well as for the molecular biology of nucleic acid.
Departamento de Microbiologia, Centro de Ciências Biológicas, Universidade Estadual de Londrina, CEP 86051-900, Londrina, Paraná, Brazil.
Flávio
F
Lauretti
Fernando
F
Lucas de Melo
Fabrício José
FJ
Benati
Eduardo
E
de Mello Volotão
Norma
N
Santos
Rosa Elisa Carvalho
RE
Linhares
Carlos
C
Nozawa
D000165:Acridine Orange; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005243:Feces / Q000821:virology; D006801:Humans; D012329:RNA, Bacterial / Q000032:analysis*; D012330:RNA, Double-Stranded / Q000032:analysis; D012401:Rotavirus / Q000235:genetics* / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000821:virology; D013194:Staining and Labeling / Q000379:methods*; D014773:Virology / Q000379:methods
D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; D000165:Acridine Orange