Small Hairpin RNA Synthesis And Its Silence Effect On Telomerase Gene Expression In Hela Cells

Objective: To establish a convenient . effective . low-cost method to synthesize small hairpin RNA creating conditions for RNA interference researches. To investigate the silence effect of Hela cells' telomerase gene expression after shRNA based on human telomerase hTERT transfected into cells.Methods:We constructed a partial double-strand DNA with T7 promoter as DNA template and synthesized small hairpinRNA in-vitro using T7 RNA polymerase. After transfecting the shRNA based on telomerase hTERT into Hela cells with calcium phosphate co-precipitation procedures, we detected Hela cells viability by Trpan blue staining method. With different amount shRNAs transfected,in different time, we assayed the telomerase activity of the transfected cells with TRAP-silver staining and PCR-EIA methods.Results: The shRNA products OD260/280 was 1.957. Its yield was 7.88 u g/20 u L reaction volumn.The Hela cells' telomerase activity declined by 30-40% after transfected by the shRNA. Conclusions: The in-vitro method that partial double-strand DNA with T7Wi l.-TOipromoter sequence as template and synthesizing by T7 RNA polymerase can product high yield, excellent purity shRNA.lt is a convenient -, effective ^ low-cost method and fit for small RNA synthesis and RNA interference researches in ordinary laboratory. The shRNA based on telomerase hTERT gene 1573-1591 sequence can silence Hela cell telomerase gene expression in a detectable extent.