Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR

Abstract

Abstract. 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, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription-PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding ␤-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. 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 RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.Key words: Classical swine fever virus; reverse transcription-PCR; RNA extraction. The reverse transcription-PCR (RT-PCR) is a valuabletechnique that is increasingly being used for diagnosis of animal diseases caused by RNA viruses, including classical swine fever virus (CSFV). 8,12,13,16,18 CSFV is a member of the genus Pestivirus, which belongs to the family Flaviviridae. 7 The virus is highly infectious for swine and causes losses in swine production throughout much of the world. 9 Rapid and accurate detection of CSFV is critical for disease containment. 16 A prerequisite for the performance of RT-PCR is an efficient method for RNA extraction. Currently there are numerous methods that can be used to isolate and purify RNA, but there are few studies comparing extraction methods for samples of animal origin. The present study was designed to evaluate 6 commercially available methods for extraction of total RNA from blood and tissue samples collected from a CSFV-infected pig. These methods were based on RNAqueous kit a (abbreviated as the RNAqueous in this report), Micro-to-midi total RNA purification system b (the Micro-tomidi), NucleoSpin RNA II c (the NucleoSpin), RNeasy mini kit d (the RNeasy), GenElute mammalian total RNA kit e (the GenElute), and TRIzol LS reagent b (the TRIzol). This article describes the results of the evaluation focusing mainly on the yield and purity of RNA extracted by these methods and the performance of the RNA in a real-time RT-PCR and a conventional RT-PCR for the CSFV genome and a conventional RT-PCR for the endogenous mammalian gene encoding cytoskeletal ␤-actin. A young pig (about 50 pounds) was inoculated intranasally with 1 ml of a highly vir...

A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.)

“…All the samples showed amplification with normal Ct values with the standard deviations in the range that allowed for meaningful analysis of expression data. The difference in the Ct value between the samples could be related to the differences in the purity of total RNA (Deng et al 2005). The expression was observed in reverse transcription negative control after 37 cycles in RNA isolated by Ghawana et al 2011.…”

Comparison of various commercial products for phenol-guanidine-based classical swine fever virus RNA extraction

“…Whereas numerous studies have been published which compare different RNA extraction procedures, both for cellular (Ruettger et al, 2010;Kong et al, 2006) and for viral (Deng et al, 2005;Guarino et al, 1997;Scheibner et al, 2000) RNA extraction, no published data are available on the comparison of various commercial products that are all based on the phenol-guanidine principle. In the present study we compare the performance of TRIzol ® with several other brand name products based on the same principle for the extraction of CSFV RNA from cell culture supernatant, serum and tonsils.…”

Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation

“…The procedures for these 3 methods are summarized in the schematics in Figure 2. Some studies have suggested the yield of RNA obtained using the GITC-based method can be higher than the column based method for some samples [22][23][24][25].…”