Efficient siRNA Transfection and shRNA Lentiviral Delivery

For siRNA transfection cells were plated at a density of 50–80% on 6-cm dishes 1 d before transfection. siRNAs were purchased from Dharmacon and were used at a final concentration of 20 nM. Transfection was carried out using the Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. Lentiviral vector plasmids for stable, constitutive or inducible expression of secreted luciferases GLuc and CLuc coupled to p53 or nontargeting control shRNAs were described previously29 (link). shRNA sequences from Open Biosystems are as follows: shFancD2.2 (V2LHS_139155), shFancD2.3 (V3LHS_383034), shFancD2.4 (V3LHS_383035). siRNA sequences: siFancD2.6 CAACATACCTCGACTCATT, siFancD2.8 GGAGATTGATGGT CTACTA, simTOR.8 GGCCAUAGCUAGCCUCAUA, simTOR.9 CAAAGGA CUUCGCCCAUAA, simTOR.10:GCAGAAUUGUCAAGGGAUA, simTOR.11 CCAAAGCACUACACUACAA.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Wanzel M., Vischedyk J.B., Gittler M.P., Gremke N., Seiz J.R., Hefter M., Noack M., Savai R., Mernberger M., Charles J.P., Schneikert J., Bretz A.C., Nist A, & Stiewe T. (2015). CRISPR-Cas9-based target validation for p53-reactivating model compounds. Nature chemical biology, 12(1), 22-28.

Publication 2015

Lipofectamine RNAiMAX reagent shRNA shFancD2.4 (V3LHS_383035

Cells Dishes Lipofectamine Luciferases Plasmids Shrna Sirna Transfection Vector

Corresponding Organization :

Other organizations : Philipps University of Marburg, Universities of Giessen and Marburg Lung Center, German Center for Lung Research

Top 5 similar protocols

Variable analysis

independent variables

SiRNA transfection
Lentiviral vector plasmids for stable, constitutive or inducible expression of secreted luciferases GLuc and CLuc coupled to p53 or nontargeting control shRNAs

dependent variables

Secreted luciferase activity (GLuc and CLuc)

control variables

Cell density (50-80%) on 6-cm dishes 1 day before transfection
SiRNA concentration (20 nM)
Transfection reagent (Lipofectamine RNAiMAX)

positive controls

Nontargeting control shRNAs

negative controls

P53 shRNAs

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!