Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region
Fig 7
pRNA mediated gene activation.
(A) Screening of three dsRNAs (100nM) targeting non-CpG sites in the pRNA of HPV18 in HeLa cells. RNA from the transfected cells was analyzed by Real time PCR 72 hours post transfection. Expression ratio was calculated with respect to 18S, POLR2A, PPIA andβ-actin by REST software. S8-dsRNA: S8 dsRNA transfected cells, S9-dsRNA: S9 dsRNA transfected cells, S10-dsRNA: S10 dsRNA transfected cells. Control: Control dsRNA transfected cells. (*) P < 0.01, (**) P = 0.03. (B) Change in bidirectional transcription after S9 dsRNA transfection. The levels of sense and antisense pRNAs were checked by transfecting Hela cells with S9 dsRNA followed by RNA isolation 72 hours post transfection. The cDNA was then analyzed by Real-Time PCR using P2 primer. P2F: cells transfected with S9 dsRNA followed by RNA isolation and reverse transcribed with P2F primer which reverse transcribes antisense pRNA, P2R: cells transfected with S9 dsRNA followed by RNA isolation and reverse transcription with P2R primer which reverse transcribes sense pRNA. Expression ratio was calculated with respect to 18S rRNA using delta-delta Ct method normalized to Control dsRNA treated samples.