We use Qiagen RNA extraction kit routinely but when cell lines infected with HCV serum and RNA was isolated by same method there were no promising results.
The Qiagen RNeasy mini kit should work fine for Huh7 or derivatives thereof. Only for HCV RNA isolations from serum or cell culture supernatants would I recommend the Qiagen viral RNA kits. Are you using JFH1 or more efficiently replicating viral genomes such as J6/JFH1 or Jc1?
Dear Sir, for extraction we are using QiaAmp viral RNA mini kit. We are not using any clone like JHF1 etc
We simply transfected Huh7 by adding serum of HCV positive patient about 500 ul with viral load of 10,000,000 copies/ml.
We isolated 1 ml cells & supernatant from culture after 48 hours of incubation and centrifuged it for 30 minutes. we then used 140 ul of pellet cum supernatant material for RNA isolation.
we are following this paper
Development of persistent HCV genotype 3a infection cell culture model in huh-7 cell
Whenever attempting to launch HCV non-JFH1 in Huh7 cells I would recommend adding a positive control of JFH1. Thus far, only a hand full of genomes were successfully and reproducibly launched in Huh7 cells. Patient serum itself may contain large quantities of inhibitory factors such as neutralizing antibodies and other thus far unknown restriction factors.
The Huh7.5 cells have been selected to be permissive to the JFH-1 clone of HCV. To date, they haven't been useful for studying HCV from patient sera. It may be that in selecting the permissive cells they made them lose proteins/factors that let them be infected by more normal HCV.
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