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J Vet Diagn Invest 17:574–578 (2005)Comparison of six RNA extraction methods for the detection of classical swine fevervirus by real-time and conventional reverse transcription–PCRMing Y. Deng,1 He Wang, Gordon B. Ward, Tammy R. Beckham, Thomas S. McKennaAbstract. Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system,NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, wereevaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription–PCR (RTPCR)for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positiveresults in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encodingb-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCRassays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methodsare more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood andtissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures.For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueouskit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElutemammalian total RNA kit was most likely to be free of contaminations with genomic DNA. |
