Comparison of RNA isolation methods for detection of classical swine fever virus from tissue samples

The present study was aimed to evaluate three RNA isolation strategies specifically TRIzol® reagent (Sigma-Aldrich) method, RNeasy® mini kit (Qiagen) and High pure RNA tissue kit (Roche) for detection of classical swine fever virus (CSFV) from different tissues of domestic pig origin stored in different conditions namely, fresh, frozen, lyophilised and formalin fixed. The purification methods were compared for yield of total RNA, purity and presence of polymerase chain reaction (PCR) inhibitors. TRIzol® reagent method produced the highest RNA yield whereas the purity was found to be superior with RNeasy® mini kit when compared with other two methods. The reverse transcription polymerase chain reaction (RT-PCR) targeting 5’ UTR, yielded specific amplification of 421 bp amplicon from all the samples, with varied band intensities among different tissue types, tissue groups and method of purification. It is concluded that RNA purified by all the three methods can be used for downstream detection of CSFV by RT-PCR from different tissues. However, High pure RNA tissue kit showed better amplification intensities in all groups of samples with appreciable intensity difference for formalin-fixed samples among the three methods.