MiniVectors to Deliver RNA

mv-H1-shRNA/eGFP: Sent 10ug sample at 142 ng/µl in H2O

Experiment courtesy of:
Sandesh Subramanya, Ph.D
Senior Scientist at Bioo Scientific Corporation

MiniVector encoding shRNA against eGFP was transfected into two GFP expressing cell lines. Knockdown of eGFP expression was evaluated by flow cytometry at 72 hours post-transfection.

Methods

Two cell lines were provided that had been modified to stably express eGFP. The H1299 cell line (lung adenocarcinoma, NCI-H1299) had been selected by FACs, resulting in a more homogeneous and highly expressing cell population. The HeLa cell line (human cervical adenocarcinoma) had not been selected by FACs and was highly heterogenous so that only a small portion showed strong eGFP expression. MiniVector encoding an shRNA against eGFP was complexed with Bioo Scientific’s Nextfect™ 2012 transfection agent. Three mass amounts of DNA were tested: 0.3 µg, 0.6 µg, and 1.0 µg of DNA to 2×10^5 cells in a 96 well flat bottom plate. Control sample with Nextfect™ 2012 transfection reagent alone was also tested. Cells were evaluated by flowcytometry at72 hours post transfection.

Results

MiniVector

Discussion

For both cell lines, only the highly expressing portion of the cell population was used for determining the efficiency of knockdown. For the H1299 cell line, MiniVector showed 80% knockdown of eGFP expression for the 1.0 µg and 0.6 µg dose and about 60% knockdown for the 0.3 µg dose. HeLa cell line showed about 63% knockdown for the 1.0 µg dose, 60% knockdown for the 0.6 µg dose, and 58% knockdown for the 0.3µg dose.