Long-term gene silencing in mammalian cells using siRNA

W W W . Q I A G E N . C O M

Frank Narz, Silke Janhsen, and Martin Weber

QIAGEN GmbH, Hilden, Germany

IntroductionGene silencing using siRNA-mediated RNAi has become a powerful tool in cell biology, addressing a range ofresearch 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 protein is also knocked down.

This knockdown is transient, especially when working with proliferating cell lines where the intracellular siRNA isdiluted with each cell division. As most cell lines have to be subcultured approximately twice a week, siRNA-mediated RNAi experiments are usually analyzed 3–4 days after transfection. In many cases, this short-term knockdown is too brief and phenotypic effects that require a longer duration of knockdown of the target protein cannot be examined.

We have developed a protocol that allows gene silencing in mammalian cells for more than 2 weeks. siRNA is transfected using lipid-based HiPerFect Transfection Reagent every time the cells are diluted and plated into a newculture plate.

RNAi Leads to Transient Knockdown Protocol for long-term silencing using HiPerFect Reagent

Long-term silencing in HeLa and MCF-7 cells Long-term silencing does not affect cell viability� Transfection using 5 nM MAPK1 siRNA and HiPerFect Transfection Reagent each time cells were split resulted

in prolonged knockdown of the target mRNA (Figure 2).

� When cells were transfected only once, repeated splitting cycles resulted in loss of the silencing effect (Figure 2).

� Cell viability was assessed by staining cells with Calcein AM dye after either a single transfection or after 3 transfections. Calcein AM fluoresces green in living cells.

� Calcein AM staining shows that repeated siRNA transfection using HiPerFect Transfection Reagent does notaffect cell viability (Figure 3).

� Long-term MAPK1 silencing in HeLa cells resulted in reduced cell numbers, probably caused by reduced proliferation.

� The same samples were used for gene expression analysis and results showed that repeated transfections resulted in prolonged silencing (Figure 4).

Summary of results

Addition of siRNA–HiPerFect Reagent complexes after cells are split allows long-term knockdown. The detailed protocol is available from QIAGEN Technical Services.

Effective MAPK1 Silencing after Multiple Splitting Cycles

Viable Cells after Repeated Transfections

HeLa MCF-7

Repeated transfection prolongs silencing without affectingviability

Choice of transfection reagent affects viability� HeLa cells were transfected with MAPK1 siRNA

using HiPerFect Transfection Reagent, Reagent L, orReagent O. The siRNA concentrations used werebased on the manufacturers’ instructions: 5 nM withHiPerFect Reagent; 33 nM with Reagent L; 106 nMwith Reagent O (Figure 5).

� HiPerFect Reagent allowed the use of the lowestsiRNA concentration (5 nM) and cells were healthyand viable after transfection.

� Reagent L required the use of a higher siRNA con-centration (33 nM) and toxicity was evident afterrepeated transfection. Cells transfected with ReagentO did not show cytotoxicity, but this reagent required the use of the highest siRNA concentration(106 nM).

� Repeated siRNA transfection using HiPerFectReagent results in prolonged silencing (Figure 4).

� The viability of the cells in these experiments was notaffected by repeated siRNA transfection (Figure 3).

Melanin synthesis is a candidate model system requiringlong-term silencing

Silencing of the melanin synthesis key enzyme tyrosinase

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� The newly developed transfection protocol allows gene knockdown in mammalian cells for over 2 weeks.

� The protocol involves siRNA delivery using HiPerFect Transfection Reagent every time the cells are diluted andreplated.

� The low cytotoxicity of HiPerFect Transfection Reagent, in combination with its suitability for transfection of lowsiRNA amounts, allows repeated siRNA transfection without affecting cell viability.

� Data presented here show long-term silencing of MAPK1 and lamin A/C in HeLa and MCF-7 cells using various siRNA concentrations (Figures 2 and 4).

� Viability staining showed that cells were healthy after repeated transfections when HiPerFect TransfectionReagent was used (Figures 3 and 5).

� 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).

� When siRNA is delivered into the cell, it meditatescleavage of homologous mRNA and so translationis prevented (see flowchart).

� mRNA degradation leads to knockdown of the target protein. However the effect is transient andgene expression is restored as siRNAs are dilutedwith each cell division and if cell cultures are split(Figure 1).

Prepare complexes

Apply to cellsIncubateChange medium after 6–24 h

When cells are confluent:Remove mediumWash cellsAdd Trypsin/EDTA

Add mediumTransfer to a new plate

Add complexesIncubateChange medium after 6–24 h

Prepare complexes

Figure 2 HeLa or MCF-7 cells (8 x 104) were plated in 24-well plates and transfected with 5 nM MAPK1 siRNA. After 48 hours, control cultures that had not been splitwere lysed and RNA was purified using the RNeasy® Mini Kit or the RNeasy 96 Kit, followed by gene expression analysis using the gene-specific TaqMan® probe and primersand the QuantiTect® Probe RT-PCR Kit. The expression level of MAPK1 was normalized to that of GAPDH. The remaining cultures were split twice a week and either retransfected at each split or replated without transfection. After 18 days, MAPK1 expression was determined by quantitative, real-time RT-PCR as described.

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Figure 3 HeLa cells (8 x 104) were plated in 24-well plates and transfected with 20 nM siRNA targeting MAPK1, GFP (nonsilencing siRNA), or lamin A/C. Mocktransfected cells (HiPerFect Reagent only) were also examined. Cultures were split twice a week and either retransfected at each split or replated without transfection.After 9 days and 2 splitting cycles, viable cells were stained using Calcein AM. These cells were also used for gene expression analysis by quantitative, real-time RT-PCR (see Figure 4).

� B16-F1 is a murine melanocytic cell line that synthesizes melanin. Melanin is formed in intracellularvesicle-like structures and is secreted into the medium (Figure 6).

� Melanin is synthesized from tyrosine and tyrosinaseis the rate-limiting enzyme (see flowchart).

� To generate B16-F1 cells that lack melanin, long-term silencing of tyrosinase is required becausemelanin is still present for some time after tyrosinaseis downregulated.

� B16-F1 cells were transfected with each one of 2 HP GenomeWide siRNAs targeting tyrosinase orwith lamin A/C siRNA as a negative control. HP GenomeWide siRNAs are available at theGeneGlobe™ Web portal ( www.qiagen.com/GeneGlobe ).

� Tyrosinase knockdown was analyzed by westernblotting. After 72 hours, tyrosinase was effectivelyknocked down at the protein level using eachsiRNA (Figure 7).

� Melanin secretion into the medium was monitoredspectrophotometrically (Figure 8). Even after tyrosinaseknockdown at 72 hours after transfection, melaninsecretion into the medium has not yet decreased.

B16-F1 Cells Secrete Melanin into the Medium

Tyrosinase Knockdown at the Protein Level

Melanin Still Present in the Medium afterTyrosinase Knockdown

Melanin Synthesis Pathways

ProteinRNA

~48h ~96h ~120h

Transfection

RNAi Mechanism

Long-Term Gene Silencing Procedure

Transient mRNA and Protein Knockdown

Figure 1 siRNA transfection leads to mRNA and subsequently protein knockdown,but the effects are short-lived.

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Prolonged Knockdown after Repeated Transfections

Cell Viability Using Different Transfection Reagents

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Figure 4 HeLa cells were transfected with 20 nM siRNA targeting MAPK1 or lamin A/C. siRNA targeting GFP (nonsilencing siRNA) was also transfected.Mock transfected cells were also analyzed. After 48 hours, control cultures that had not been split were used for quantitative, real-time RT-PCR analysis. Cultures weresplit twice a week and either retransfected at each split or replated without transfection. After 9 days and 2 splitting cycles, MAPK1 and lamin A/C expression wasdetermined by quantitative, real-time RT-PCR. Cell viability of these cultures was also assessed (Figure 3).

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Figure 6 B16-F1 cells synthesize melanin, which is then secreted into the medium, causing the medium to become brown.

Figure 7 B16-F1 cells (8 x 104) were plated in 24-well plates and transfectedusing 5 nM or 20 nM siRNA and HiPerFect Reagent. Two siRNAs targeting tyrosinase were transfected. At the time points indicated, cell lysates were analyzed by western blotting using tyrosinase-specific antibodies. Blots werealso probed with tubulin-specific antibody as an internal control. Figure 8 Cells were transfected as described in Figure 7. Spectrophotometric

analysis showed that melanin was still present in the medium 72 hours aftertransfection.

Figure 5 HeLa cells (8 x 104) were plated in 24-well plates and transfected using5 nM MAPK1 siRNA and HiPerFect Reagent, 33 nM MAPK1siRNA andReagent L, or 106 nM MAPK1 siRNA and Reagent O. siRNA concentrationswere recommended by the manufacturer. Cultures were split twice a week andeither retransfected at each split or replated without transfection. After 9 days,cell viability was examined by phase contrast microscopy.

HiPerFect Reagent L Reagent O

One transfectionSplit twice

Three transfectionsSplit twice

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, eitherby 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. andF. Hoffmann-La Roche Ltd.

© 2005 QIAGEN, all rights reserved.

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