1032295_0805_SPOS_ELSO_Meeting.qxd 12.08.2005 18:29 Uhr Seite 1
Long-term gene silencing in mammalian cells using siRNA
Frank Narz, Silke Janhsen, and Martin Weber QIAGEN GmbH, Hilden, Germany
Introduction Summary of results
Gene silencing using siRNA-mediated RNAi has become a powerful tool in cell biology, addressing a range of The newly developed transfection protocol allows gene knockdown in mammalian cells for over 2 weeks. research questions in functional genomics and drug discovery. After delivery of siRNA into the cell, rapid degradation of the target mRNA can be detected, often within 24 hours, and subsequently the corresponding The protocol involves siRNA delivery using HiPerFect Transfection Reagent every time the cells are diluted and protein is also knocked down. replated. This knockdown is transient, especially when working with proliferating cell lines where the intracellular siRNA is The low cytotoxicity of HiPerFect Transfection Reagent, in combination with its suitability for transfection of low diluted with each cell division. As most cell lines have to be subcultured approximately twice a week, siRNA amounts, allows repeated siRNA transfection without affecting cell viability. siRNA-mediated RNAi experiments are usually analyzed 3–4 days after transfection. In many cases, this Data presented here show long-term silencing of MAPK1 and lamin A/C in HeLa and MCF-7 cells using short-term knockdown is too brief and phenotypic effects that require a longer duration of knockdown of the various siRNA concentrations (Figures 2 and 4). target protein cannot be examined. Viability staining showed that cells were healthy after repeated transfections when HiPerFect Transfection We have developed a protocol that allows gene silencing in mammalian cells for more than 2 weeks. siRNA is Reagent was used (Figures 3 and 5). transfected using lipid-based HiPerFect Transfection Reagent every time the cells are diluted and plated into a new culture plate. A phenotypic assay analyzing melanin synthesis and secretion after tyrosinase knockdown identifies this pathway as a model system where long-term silencing is necessary for melanin research (Figures 6, 7, and 8).
Trademarks: QIAGEN®, GeneGlobe™, QuantiTect®, RNeasy® (QIAGEN Group); TaqMan® (Roche Group). siRNA technology licensed to QIAGEN is covered by various patent applications, owned by the Massachusetts Institute of Technology, Cambridge, MA, USA and others. Purchase of QIAGEN products for PCR containing HotStarTaq DNA Polymerase is accompanied by a limited license to use them in the polymerase chain reaction (PCR) process for research and development activities in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Applied Biosystems or as purchased, i.e. an authorized thermal cycler. The PCR process is covered by the foreign counterparts of U.S. Patents Nos. 4,683,202 and 4,683,195 owned by F. Hoffmann-La Roche Ltd. The 5' nuclease process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. © 2005 QIAGEN, all rights reserved.
RNAi Leads to Transient Knockdown Protocol for long-term silencing using HiPerFect Reagent When siRNA is delivered into the cell, it meditates RNAi Mechanism Addition of siRNA–HiPerFect Reagent complexes after cells are split allows long-term knockdown. The detailed cleavage of homologous mRNA and so translation protocol is available from QIAGEN Technical Services. is prevented (see flowchart). Synthetic siRNA (21–23 nt dsRNA) Long-Term Gene Silencing Procedure mRNA degradation leads to knockdown of the RISC Assembly target protein. However the effect is transient and gene expression is restored as siRNAs are diluted Prepare complexes with each cell division and if cell cultures are split
(Figure 1). RNA unwound single-stranded siRNA incorporated into RISC Apply to cells Transient mRNA and Protein Knockdown Incubate Change medium after 6–24 h Transfection
mRNA target When cells are confluent: recognition Remove medium Wash cells Add Trypsin/EDTA
Protein RNA
Add medium Prepare complexes mRNA cleavage Transfer to a new plate ~48h ~96h ~120h
Figure 1 siRNA transfection leads to mRNA and subsequently protein knockdown, Add complexes but the effects are short-lived. Incubate Change medium after 6–24 h
Long-term silencing in HeLa and MCF-7 cells Long-term silencing does not affect cell viability