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#1 Faiz Ahmed Raza

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Posted 20 February 2019 - 05:12 AM

I am working on the dengue virus serotyping (Lanciotti et al 1992) using NS1 positive human blood samples. However the samples are degraded and whenever I try to isolate viral RNA, I always get lots of human RNA i.e. I always get band with random primer mix or oligo primer but not with gene specific primer. One of my colleague suggested to use RNAse A before starting viral RNA extraction to remove contaminating human RNA. RNAse A will not affect viral RNA because it will remain protected in the nucleocapsid. However I am reluctant to try RNAse A as they are considered as very sturdy enzymes. Few also suggested using disposable 0.4um filter assembly to remove debris to improve the efficiency. I am bit inclined to use filtration assembly but not RNAse A. Kindly share your experience in this regard especially about downstream deactivation of RNAse A. 



#2 bob1

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Posted 22 February 2019 - 12:16 PM

RNA extraction kits are pretty good at removing most of the proteins in a solution. You can also get a number of enzymes (e.g. murine RNase inhibitor) and other things that will inhibit or stop RNase activitiy. You can definitely do as your colleague suggested, however you would need to play around with the conditions to suit your work-flow and extraction methods.

 

If the samples are degraded before the you get to do a RNA extraction, you might find that RNase treatment also damages RNA that is present from degraded viral particles - resulting in false negatives. One way around this would be to use an enrichment system, such as having long dengue sequence-specific oligos bound to beads, which then can be used in an extraction.






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