Abstract
TRIzol ® , TRI Reagent ® , and RNAzol ® are widely used commercial reagents for the extraction of cellular or viral RNA. Several other brand name products, some of which are advertised for the processing of specific sample types such as blood, are also available. Here, we compare the efficiency of these products for classical swine fever virus RNA extraction from cell culture supernatant, serum, and tonsil tissue, assessed by quantitative RT-PCR. Furthermore, the detection of a synthetic RNA transcript used as an internal positive control for extraction and RT-qCR was compared as well. Most tested products showed a similar extraction efficiency, and none of the products recommended for specific sample types performed better than the all-purpose reagents. We also show that the homogenization method for tissue samples has a significant impact on the detection efficiency of the RNA after extraction from the homogenized tissue. Homogenization of 100 mg tissue in 5 ml cell culture medium and using an UltraTurrax ® tissue grinder yielded the best results, whereas TissueLyser ® -mediated homogenization in 1 ml cell culture medium or direct homogenization in RNA extraction medium proved to be less efficient. Keywords: RNA extraction, homogenization, comparison, TRIzol, TRI reagent, reverse transcription-quantitative PCR (RT-qPCR) African Journal of Biotechnology , Vol 13(42) 4100-4104
Highlights
Com mparis son of variou v s com mmercia al prod ducts ffor pheno ol-gua anidine e-based d class sical s swine ffever v virus
Classical swine fever (CSF) is one of the most devastating pig diseases worldwide (Penrith et al, 2011). It is caused by CSF virus (CSFV) which belongs to the genus pestivirus within the family Flaviviridae
Extraction, due to the different volumes used for homogenization
Summary
A. Several other o brand name n products, some off which are advertised ffor the proce essing of specific sa ample types such as blo ood, are als so available. M. Most tested products showed a similar s extra action efficie ency, and none of the p products rec commended d for specific c sample types perfo ormed better than the all-purpose reagents. We allso show tha at the homog genization method for tissue samp ples has a significant im mpact on the detection effficiency of tthe RNA afte er extraction from the homogenized tissue. One o e of the mostt widely used methods is the isolation of total cellular or viiral RNA by TRIzol® whicch is a mono ophasiic solution off phenol and guanidine issothiocyanate e
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