Dear Researcher,
I am doing HBV DNA transfection to Huh7 and than do experiments RTpcr to measure pgRNA , from that I observe the HBV replicate in cell. But my Prof said how do you know that come from HBV replicate but not come from the plasmid.
She said after transfection, plasmid still there in the cell and can produce pgRNA, from that pgRNA, cell will produce a complete HBV and only pgRNA from HBV should be counted, not from the plasmid.
I read so many paper but I can not find and paper recommend about this issue.
Did anyone have this problem before? Please help me
Thank you so much
Hi,
I have down some experiments like yours, transfect plasmid into Huh 7 cells and then collect cells in buffer RLT for RNA extraction and genomic / subgenomic analysis. After RNA extraction, we usually treat RNA with DNase I to eliminate any remaining of DNA.
Hope it helps you.
Li
Kou Hayakawa
Thank you so much Professor Kou, I will read carefully your answerPaniz Shirmast
Sorry for my knowledge limited, but in my understand, transfection means we add a gene of virus in plasmid and make it go inside the cell, I dont know that we can transfect a real virus...Li Wang Thanks for your answer, but HBV is a unique virus because it can reverse transcript. DNA from plasmid (include HBV gene) can transcript a pgRNA and that not the one we want to measure, we only want to measure the one replicate inside (when the HBV DNA become genome inside nucleus)
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