A comparison of two RNA isolation methods for double-stranded RNA of infectious bursal disease virus.
Infectious bursal disease virus (IBDV), a member of the birnaviridae family, contains a bisegmented double-stranded RNA (dsRNA) genome. The segments are linked covalently at 5' termini by a large (90 kDa) viral genomic protein that migrates similar to viral RNA dependent RNA polymerase of IBDV. A proteinase K digestion based approach and acid-guanidium-phenol-chloroform (AGPC) RNA extraction method were used to extract dsRNA of IBDV from infected bursae. After extraction, dsRNA of IBDV was purified by precipitation with lithium chloride. The yield and purity of dsRNA of IBDV extracted by AGPC method was lower than that of proteinase K digestion based approach. This observation correlates with the presence of a genome-linked protein in IBDV. Although dsRNA obtained by both methods are suitable for reverse transcription-polymerase chain reaction (RT-PCR) amplification of at least up to 1201 base pairs (bp) of cDNA, dsRNA extracted by the proteinase K digestion method is more suitable than that by AGPC method for the amplification of longer fragments (1958 bp) of IBDV cDNA by PCR.
Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-1175, USA.
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D000818:Animals; D018175:Birnaviridae Infections / Q000662:veterinary* / Q000821:virology; D002061:Bursa, Synovial / Q000821:virology; D002645:Chickens / Q000821:virology; D002725:Chloroform; D019286:Endopeptidase K / Q000378:metabolism; D006146:Guanidines; D007243:Infectious bursal disease virus / Q000235:genetics*; D010636:Phenols; D011201:Poultry Diseases / Q000821:virology*; D012330:RNA, Double-Stranded / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013861:Thiocyanates
D006146:Guanidines; D010636:Phenols; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D013861:Thiocyanates; C054436:guanidine thiocyanate; D002725:Chloroform; D019286:Endopeptidase K