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Highly efficient reverse transfection with siRNA in multiple wells of microtiter plates
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 104, No. 4, 329–333. 2007 DOI: 10.1263/jbb.104.329
© 2007, The Society for Biotechnology, Japan
Highly Efficient Reverse Transfection with siRNA in Multiple Wells of Microtiter Plates Satoshi Fujita,1,2 Eiji Ota,1 Chie Sasaki,1 Kota Takano,1 Masato Miyake,1* and Jun Miyake1,2 Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), 2-41-6 Aomi, Koto-ku, Tokyo 135-0064, Japan1 and Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan2 Received 27 February 2007/Accepted 20 July 2007
We have developed an efficient and inexpensive method of reverse transfection from the solid phase to suppress genes with siRNA. The method enabled the realization of (i) a high efficiency of transfection; (ii) transfection of various types of cell; (iii) a high efficiency of gene knockdown by siRNA; (iv) a low toxicity to cells; and (v) a long-term stabilization (more than 210 d) of attached transfection mixture including siRNA in multiple wells. Although array-based reverse transfection has advantages in terms of miniaturization, the method has the advantage of enabling the inclusion of various soluble factors, such as humoral factors, drugs and ligands that affect gene expression, because the liquid phase is partitioned within the individual wells of each microtiter plate. Our method of reverse transfection with siRNA in multiple wells is a powerful and high-throughput tool for the analysis of signaling pathways. [Key words: reverse transfection, solid phase, transfection with siRNA, lines of human cells, high-throughput]
it is expected that reverse transfection from the solid phase using microtiter plates might lead to economy on the costly reagents that are required for transfection and to the simplification of the process of transfection. In addition, it is easy to isolate cellular components, such as proteins and mRNAs, from individual wells. One of the important points for realizing reverse transfection in multiple wells of a microtiter plate is the uniformity of transfection of cells. In published methods of reverse transfection from the solid phase, transfection of cells tends to occur in the solid-phase region of reagents, yielding a non uniform efficiency of transfection of cells in various regions of a well. By contrast, in conventional transfection or reverse transfection from the liquid phase, large amounts of costly reagents are necessary even though a uniform transfection is expected (7–10). Moreover, a long-term stabilization of reagent mixtures that include DNA or siRNA on the solid phase is necessary for storing microtiter plates whose wells had been precoated. In this study, we developed a method of reverse transfection of cells with siRNA in wells of microtiter plates. We successfully transfected both uniformly and at a high efficiency various lines of cells, derived from the cervix, breast, and kidney, for example, using siRNA. Our method also enabled a rapid concurrent analysis, which is laborious when conventional transfection is performed. Our method is economical and the reagent mixture remained stable in the wells of microtiter plates for at least 210 d.
Methods for reverse transfection from a solid phase, whereby plasmid DNA or siRNA on the solid phase are introduced into adherent cells that have been seeded on a contact surface, have been developed by several groups since Ziauddin and Sabatini (1) first reported such a method (1– 6). In our laboratory, we have used fibronectin in an effort to generalize the process of retention of DNA or siRNA and to promote transfection. Both DNA and siRNA were successfully introduced into various cell lines, such as HEK293 cells, HeLa cells, NIH3T3 cells, HepG2 cells, and human mesenchymal stem cells (hMSCs) (4). This method is mainly used for the preparation of cell microarrays for gene transfection, and we call such microarrays transfection microarrays (4). They have enabled the miniaturization and simplication of a method of screening of gene function, identification of drug targets, and the characterization of signal transduction by high-throughput analysis. On the other hand, a method using multiple wells on microtiter plates should be developed. Although reverse transfection with genes from the solid phase on wells is not superior to that on a microarray chip in terms of miniaturization, the former method enables the inclusion of soluble factors, such as humoral factors, drugs and ligands, and the analysis of their effects on transfectants in individual wells because the liquid phase is partitioned by the wells of the microtiter plate. Moreover, * Corresponding author. e-mail: [email protected] phone: +81-(0)3-3599-8941 fax: +81-(0)3-3599-8949 The first two authors contributed equally to this work. Abbreviations: hMSCs, human mesenchymal stem cells; PVA, polyvinyl alcohol; QPCR, quantitative polymerase chain reaction. 329
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MATERIALS AND METHODS All cells were obtained from commercial sources and cultivated in an incubator at 37°C in an atmosphere of 5% CO2 in air. Human MSCs (Cambrex BioScience, Walkersville, MD, USA) were used after fewer than five passages to avoid phenotypic changes. Cy3labeled siRNAs (Ambion, Austin, TX, USA) were used for assays of transfection efficiency, and conventional siRNAs (Qiagen, Valencia, CA, USA) were used for investigations of gene-knockdown efficiencies of siRNAs in transfected cells and of the stability of the siRNA mixture. We precoated each well of a microtiter plate for the reverse transfection of genes from the solid phase. We placed 40 µl of a mixture (1: 1, v/v) of 70% EtOH and a solution of dextran (MW 40,000; Wako, Osaka; 45 g/l in phosphate-buffered saline, PBS) in the center of each well of a 24-well microtiter plate and then dried the plate in a vacuum chamber for 1 h. We mixed 7 µl of a 20 µM solution of siRNA in distilled water with 16 µl of the solution of dextran and 2 µl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and incubated the mixture at room temperature for 20 min. Then we added 92 µl of a 0.5% solution of polyvinyl alcohol (PVA; MW 500; Wako) in distilled water. An appropriate volume of the mixture (4 µl per spot in a 96-well plate or 22 µl per spot in a 24-well plate) was spotted in each well of a microtiter plate. When all the wells had been similarly treated, the plate was dried in a vacuum chamber for 1 h. To investigate transfection efficiencies and gene-knockdown efficiencies of siRNAs, we placed cells (0.4–2 ×105 cell per well in a 24-well format) in an appropriate medium (DMEM with 10% FBS or recommended medium by supplier) in each well for reverse transfection. In the case of a conventional transfection method, siRNA was transfected into cells using Lipofectamine 2000 in accordance the supplied protocol. After 24 h, we obtained phase-contrast and fluorescence images of cells in each well with a fluorescence microscope (IX81; Olympus, Tokyo). For quantitative polymerase chain reaction (QPCR) analysis, we extracted mRNAs from cells in each well using a TurboCapture mRNA kit (Qiagen) and performed QPCR using a One Step SYBR PrimeScript RTPCR kit (Takara Bio, Shiga) and an ABI PRISM 7700 system (Applied Biosystems, Foster City, CA, USA) in accordance with the instructions of the manufacturers. The relative efficiency of the
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knockdown of the expression of the gene for CDK2 by anti-CDK2 siRNA was calculated against the control with a non target siRNA. Cytotoxicity induced by reverse transfection from a solid phase was confirmed by staining of dead cells with EthD-1 (Invitrogen). After 24 h of reverse transfection of each cell strain with siRNA, 5 µM EthD-1 in DMEM was added to cells and the cells were incubated for 30 min. Treated cells were washed twice with PBS and after 10 min, dead cells were detected on the basis of the fluorescence of EthD-1. As a control experiment, dead cells were prepared by adding 70% MeOH before staining with EthD-1 (Fig. 2). To investigate the stability of the siRNA mixture in wells of microtiter plates, individual plates, after drying in the vacuum chamber as described above, were vacuum-packed with a desiccant and stored at 4°C.
RESULTS AND DISCUSSION We first investigated the transfection efficiency of our reverse transfection method using siRNA and the toxicity to various cell lines as induced by the method. Figure 1 shows the results for three cell lines (HeLa, SK-BR-3 and MCF7), which were transfected with Cy3-labeled non target siRNA. The transfection efficiency and the localization of siRNA can be assessed from the fluorescent signals. In these cell lines, siRNA was introduced at a high efficiency and was localized in the cytoplasm, which is a prerequisite for the knockdown of mRNA because siRNA generally acts in the cytoplasm. Moreover, cytotoxicity induced by reverse transfection from the solid phase was confirmed by staining dead cells with EthD-1 (Fig. 2). The number of dead cells transfected with siRNA was equal to that not transfected. The number of dead cells transfected with siRNA was markedly smaller than that treated with MeOH. The results show that the toxicity of siRNA transfection to each cell line was very low and no cell death appeared to result from the transfection procedure. Next, we investigated the uniformity of transfection. When cell arrays are used as, for example, described by Ziauddin
FIG. 1. Photographs of three typical lines of cells transfected with Cy3-labeled non target siRNA. (A) HeLa; (B) SK-BR-3; (C) MCF7; upper images, phase-contrast; center images, fluorescence due to Cy3; lower images, merged phase-contrast and fluorescence images; left side of each rectangle, 4× lens; right side of each rectangle, 20× lens. Cells were transfected with Cy3-labeled non specific siRNA in 96-well plates by the wellbased reverse-transfection method. Images were recorded after incubation for 24 h at 37°C in an atmosphere of 5% CO2 in air.
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FIG. 2. Cytotoxicity to three typical lines of cells as induced by reverse transfection from solid phase. (A) HeLa; (B) SK-BR-3; (C) MCF7; upper images, cells without transfection; center images, cells with transfection of non target siRNA; lower images, MeOH-treated cells without transfection; left side of each rectangle, phase contrast; right side of each rectangle, fluorescence staining with EthD-1. After 24 h of reverse transfection of each cell strain with siRNA, 5 µM EthD-1 in DMEM was added to cells and the cells were incubated for 30 min. The cells were washed twice with PBS and after 10 min dead cells were detected on the basis of the fluorescence of EthD-1. As a control experiment, dead cells were prepared by adding 70% MeOH to cells before staining with EthD-1.
and Sabatini (1) and us (4), the spotted solution of transfection reagents dries up immediately and uniformly, because the volume of the transfection solution is very small (10 nl– 25 nl per spot). However, in our new method, because the transfection mixture used to coat the wells does not dry up immediately, the mixture tends to aggregate on the sides of the wells as a result of surface tension, which inhibits uniform transfection. Therefore, we added polyvinyl alcohol (PVA) to our transfection mixture. PVA, which is utilized generally as a dispersant or emulsifying agent, is very strongly hydrophilic and it lowers the surface tension of aqueous solutions. Therefore, we expected that the addition of PVA would promote the uniform spreading of the transfection mixture throughout each well. We postulated that PVA is not toxic to cells because PVA is a physiologically inactive material that has been approved as a resin for food packaging by the US Food and Drug Administration (FDA). We also added dextran to the transfection mixture to enhance transfection efficiency and the spreading of the transfection solution over the entire surface of each well. It has been reported that a high concentration of sucrose in the transfection mixture contributes to the stability of the transfection reagent complex and increases transfection efficiency (11). However, a high concentration of sucrose interferes with the immobilization of the transfection complex on a solid surface; the complex spreads beyond the area of the spot upon addition of the medium in which cells are suspended and, as a result, even cells outside the area of the spot are transfected (11). Although this is major problem for arraybased reverse transfection, it is an advantage for well-based reverse transfection. We found that various polysaccharides,
FIG. 3. Images of siRNA on the lower right side of a well (A) before and (B) after well-based reverse transfection. Left images show Cy3labeled siRNA in the transfection mixture prior to cell seeding (A). Right images show Cy3-labeled siRNA and cells in the same region of the well, after cell seeding and incubation for 16 h as described in the text (B). Images were obtained at two magnifications, as indicated.
including glucose, sucrose and dextran, enhanced the efficiency of transfection (data not shown). Figure 3 shows fluoresence images of siRNAs on the lower right side of a well before and after well-based reverse transfection. The siRNA was labeled with Cy3 to allow us
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TABLE 1. Gene-knockdown efficiency of anti-CDK2 siRNA after well-based reverse transfection of various lines of cells Knockdown efficiency (%) HeLa Adenocarcinoma Cervix 97 T-47D Ductal carcinoma Mammary gland; breast 94 SK-BR-3 Adenocarcinoma Mammary gland; breast 96 MCF7 Adenocarcinoma Mammary gland; breast 97 MDA-MB-231 Adenocarcinoma Mammary gland; breast 87 MDA-MB-435S Ductal carcinoma Mammary gland; breast 86 MDA-MB-468 Adenocarcinoma Mammary gland; breast 88 HCC1428 Adenocarcinoma Mammary gland; breast 46 HCC1806 Primary acantholytic squamous cell carcinoma Mammary gland; breast 82 HCC1954 Ductal carcinoma Mammary gland; breast 92 AU565 Adenocarcinoma Mammary gland; breast 72 HEK293 – Kidney 97 HepG2 Hepatocellular carcinoma Liver 98 NT2 Malignant pluripotent embryonal carcinoma Testis 94 SHSY5Y Neuroblastoma Brain 85 hMSC – Mesenchymal stem cell 94 The relative efficiency of the knockdown of expression of the gene for CDK2 with anti-CDK2 siRNA was calculated against the control with a non target siRNA by QPCR analysis. Cell line
Source
to visualize siRNA that was attached to the solid phase before transfection and to localize it in the cytoplasm after transfection. Images on the left show Cy3-labeled siRNA in the transfection mixture prior to cell seeding. After cell seeding and incubation for 16 h, we photographed the labeled siRNA and cells in the same region of the well (Fig. 3B). As shown in Fig. 3, before reverse transfection, the attached transfection mixture, including siRNA, did not spread uniformly in each well even though PVA induced some spreading of the transfection mixture (Fig. 3A, upper left). Moreover, Cy3-labeled siRNA aggregated as plaques on the solid phase (Fig. 3A, lower left). However, after reverse transfection, the attached siRNA was present inside cells and the efficiency of transfection was uniform in all regions of the well (Fig. 3B). Conventional liquid-based transfection with liposomes, such as Lipofectamine 2000, requires 30 times the volume of transfection mixture as that used in our method. We cannot assume, therefore, that the transfection mixture becomes resuspended in the medium from the solid surface and then siRNA is introduced into cells. Two mechanisms might be involved: transfection might occur from the interface between cells and the solid mixture by phagocytosis; a density gradient formed by dextran might inhibit diffusion components of the redissolved transfection mixture. Next, we investigated the efficiency of gene knockdown in 15 lines of human cells including hMSCs (Table 1). The relative efficiency of the knockdown of the expression of the gene for CDK2 by anti-CDK2 siRNA was calculated against the control with a non target siRNA by QPCR analysis. Almost all cells were transfected with the specific siRNA (anti-CDK2) at a high efficiency. Only Hcc1428 cells derived from a human adenocarcinoma were resistant to the knockdown of the CDK2 gene (46%). Even hMSCs, which are difficult to transfect and gene knockdown by conventional methods, were transfected and the CDK gene was suppressed at a high efficiency (94%). To compare our well-based method with a conventional method, we used anti-CDK2 siRNA to transfect various cell lines by each method. After incubation
Organ (human)
FIG. 4. Comparison of the gene-knockdown efficiency of specific anti-CDK2 siRNA between well-based reverse transfection and conventional transfection. In the case of respective transfection methods, namely conventional transfection and reverse transfection, siRNA was transfected into cells using Lipofectamine 2000 in accordance the procedures shown in materials and methods. After transfection of indicated cells with anti-CDK2 siRNA by each method and incubation for 24 h, the levels of expression of the gene for CDK2 were compared as described in the text. The relative efficiency of the knockdown of expression of the gene for CDK2 with anti-CDK2 siRNA was calculated against the control with a non target siRNA by QPCR analysis.
for 24 h, we compared the relative level of expression of the gene for CDK2 with that of the control with a non target siRNA in each cell line by QPCR (Fig. 4). In all types of cell examined, gene expression was repressed more strongly by our method than by the conventional method. In particular, the level of expression of the gene for CDK2 was decreased to 3% of the normal level in both HeLa and MCF7 cells. One of the advantages of reverse transfection from the solid phase is the long-term stabilization of the reagent mixture that includes DNA or siRNA on the solid phase (12). Therefore, we examined whether it is possible to use plates that had been precoated with the transfection mixture. To
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ACKNOWLEDGMENTS This study was performed as part of “The Project for Development of Analysis Technology for Gene Functions with Cell Arrays”, which was entrusted by the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
REFERENCES
FIG. 5. Relative gene-knockdown activity of siRNA after transfection in wells of stored plates. Plates with attached transfection mixture that included control siRNA (anti-CDK2) were stored at 4°C under a vacuum. Then HeLa cells were seeded in wells of stored plates at the indicated times and gene-knockdown efficiency was evaluated in terms of the level of expression of the gene for CDK2 by QPCR analysis.
establish and evaluate a procedure for storing treated plates, we prepared plates with the transfection mixture that included the specific siRNA (anti-CDK2) and stored them at 4°C under a vacuum for 210 d. We seeded HeLa cells in wells of stored plates at intervals and evaluated the geneknockdown efficiency of the specific siRNA in terms of the expression of the gene for CDK2. Figure 5 shows the relative gene-knockdown activity of siRNA on stored plates over time. We did not detect any reduction in gene-knockdown efficiency for 210 d. Thus, the transfection mixture was extremely stable and the treated plates may be stored for a long time under easily controlled conditions. Our new system provides an inexpensive and stable tool for the high-throughput analysis of gene knockdown. Although array-based reverse transfection has advantages in terms of miniaturization, our efficient and inexpensive method has the advantage of enabling the inclusion of various soluble factors, such as humoral factors, drugs and ligands, that affect gene expression, because the liquid phase is partitioned within the individual wells of each microtiter plate.
1. Ziauddin, J. and Sabatini, D. M.: Microarrays of cells expressing defined cDNAs. Nature, 411, 107–110 (2001). 2. Kumar, R., Conklin, D. S., and Mittal, V.: High-throughput selection of effective RNAi probes for gene silencing. Genome Res., 13, 2333–2340 (2003). 3. Mousses, S., Caplen, N. J., Cornelison, R., Weaver, D., Basik, M., Hautaniemi, S., Elkahloun, A. G., Lotufo, R. A., Choudary, A., Dougherty, E. R., Suh, E., and Kallioniemi, O.: RNAi microarray analysis in cultured mammalian cells. Genome Res., 13, 2341–2347 (2003). 4. Yoshikawa, T., Uchimura, E., Kishi, M., Funeriu, D. P., Miyake, M., and Miyake, J.: Transfection microarray of human mesenchymal stem cells and on-chip siRNA gene knockdown. J. Control. Release, 96, 227–232 (2004). 5. Baghdoyan, S., Roupioz, Y., Pitaval, A., Castel, D., Khomyakova, E., Papine, A., Soussaline, F., and Gidrol, X.: Quantitative analysis of highly parallel transfection in cell microarrays. Nucleic Acids Res., 32, e77 (2004). 6. Uchimura, E., Yamada, S., Uebersax, L., Fujita, S., Miyake, M., and Miyake, J.: A method for reverse transfection using gold colloid as a nano-scaffold. J. Biosci. Bioeng., 103,101– 103 (2007). 7. Aza-Blanc, P., Cooper, C. L., Wagner, K., Batalov, S., Deveraux, Q. L., and Cooke, M. P.: Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol. Cell, 12, 627–637 (2003). 8. Wheeler, D. B., Bailey, S. N., Guertin, D. A., Carpenter, A. E., Higgins, C. O., and Sabatini, D. M.: RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells. Nat. Methods, 1, 127–132 (2004). 9. Silva, J. M., Mizuno, H., Brady, A., Lucito, R., and Hannon, G. J.: RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells. Proc. Natl. Acad. Sci. USA, 101, 6548–6552 (2004). 10. Ovcharenko, D., Jarvis, R., Hunicke-Smith, S., Kelnar, K., and Brown, D.: High-throughput RNAi screening in vitro: from cell lines to primary cells. RNA, 11, 985–993 (2005). 11. Mannherz, O., Mertens, D., Hahn, M., and Lichter, P.: Functional screening for proapoptotic genes by reverse transfection cell array technology. Genomics, 87, 665–672 (2006). 12. Reinisalo, M., Urtti, A., and Honkakoski, P.: Freeze-drying of cationic polymer DNA complexes enables their longterm storage and reverse transfection of post-mitotic cells. J. Control. Release, 110, 437–443 (2006).
© 2007, The Society for Biotechnology, Japan
Highly Efficient Reverse Transfection with siRNA in Multiple Wells of Microtiter Plates Satoshi Fujita,1,2 Eiji Ota,1 Chie Sasaki,1 Kota Takano,1 Masato Miyake,1* and Jun Miyake1,2 Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), 2-41-6 Aomi, Koto-ku, Tokyo 135-0064, Japan1 and Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan2 Received 27 February 2007/Accepted 20 July 2007
We have developed an efficient and inexpensive method of reverse transfection from the solid phase to suppress genes with siRNA. The method enabled the realization of (i) a high efficiency of transfection; (ii) transfection of various types of cell; (iii) a high efficiency of gene knockdown by siRNA; (iv) a low toxicity to cells; and (v) a long-term stabilization (more than 210 d) of attached transfection mixture including siRNA in multiple wells. Although array-based reverse transfection has advantages in terms of miniaturization, the method has the advantage of enabling the inclusion of various soluble factors, such as humoral factors, drugs and ligands that affect gene expression, because the liquid phase is partitioned within the individual wells of each microtiter plate. Our method of reverse transfection with siRNA in multiple wells is a powerful and high-throughput tool for the analysis of signaling pathways. [Key words: reverse transfection, solid phase, transfection with siRNA, lines of human cells, high-throughput]
it is expected that reverse transfection from the solid phase using microtiter plates might lead to economy on the costly reagents that are required for transfection and to the simplification of the process of transfection. In addition, it is easy to isolate cellular components, such as proteins and mRNAs, from individual wells. One of the important points for realizing reverse transfection in multiple wells of a microtiter plate is the uniformity of transfection of cells. In published methods of reverse transfection from the solid phase, transfection of cells tends to occur in the solid-phase region of reagents, yielding a non uniform efficiency of transfection of cells in various regions of a well. By contrast, in conventional transfection or reverse transfection from the liquid phase, large amounts of costly reagents are necessary even though a uniform transfection is expected (7–10). Moreover, a long-term stabilization of reagent mixtures that include DNA or siRNA on the solid phase is necessary for storing microtiter plates whose wells had been precoated. In this study, we developed a method of reverse transfection of cells with siRNA in wells of microtiter plates. We successfully transfected both uniformly and at a high efficiency various lines of cells, derived from the cervix, breast, and kidney, for example, using siRNA. Our method also enabled a rapid concurrent analysis, which is laborious when conventional transfection is performed. Our method is economical and the reagent mixture remained stable in the wells of microtiter plates for at least 210 d.
Methods for reverse transfection from a solid phase, whereby plasmid DNA or siRNA on the solid phase are introduced into adherent cells that have been seeded on a contact surface, have been developed by several groups since Ziauddin and Sabatini (1) first reported such a method (1– 6). In our laboratory, we have used fibronectin in an effort to generalize the process of retention of DNA or siRNA and to promote transfection. Both DNA and siRNA were successfully introduced into various cell lines, such as HEK293 cells, HeLa cells, NIH3T3 cells, HepG2 cells, and human mesenchymal stem cells (hMSCs) (4). This method is mainly used for the preparation of cell microarrays for gene transfection, and we call such microarrays transfection microarrays (4). They have enabled the miniaturization and simplication of a method of screening of gene function, identification of drug targets, and the characterization of signal transduction by high-throughput analysis. On the other hand, a method using multiple wells on microtiter plates should be developed. Although reverse transfection with genes from the solid phase on wells is not superior to that on a microarray chip in terms of miniaturization, the former method enables the inclusion of soluble factors, such as humoral factors, drugs and ligands, and the analysis of their effects on transfectants in individual wells because the liquid phase is partitioned by the wells of the microtiter plate. Moreover, * Corresponding author. e-mail: [email protected] phone: +81-(0)3-3599-8941 fax: +81-(0)3-3599-8949 The first two authors contributed equally to this work. Abbreviations: hMSCs, human mesenchymal stem cells; PVA, polyvinyl alcohol; QPCR, quantitative polymerase chain reaction. 329
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MATERIALS AND METHODS All cells were obtained from commercial sources and cultivated in an incubator at 37°C in an atmosphere of 5% CO2 in air. Human MSCs (Cambrex BioScience, Walkersville, MD, USA) were used after fewer than five passages to avoid phenotypic changes. Cy3labeled siRNAs (Ambion, Austin, TX, USA) were used for assays of transfection efficiency, and conventional siRNAs (Qiagen, Valencia, CA, USA) were used for investigations of gene-knockdown efficiencies of siRNAs in transfected cells and of the stability of the siRNA mixture. We precoated each well of a microtiter plate for the reverse transfection of genes from the solid phase. We placed 40 µl of a mixture (1: 1, v/v) of 70% EtOH and a solution of dextran (MW 40,000; Wako, Osaka; 45 g/l in phosphate-buffered saline, PBS) in the center of each well of a 24-well microtiter plate and then dried the plate in a vacuum chamber for 1 h. We mixed 7 µl of a 20 µM solution of siRNA in distilled water with 16 µl of the solution of dextran and 2 µl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and incubated the mixture at room temperature for 20 min. Then we added 92 µl of a 0.5% solution of polyvinyl alcohol (PVA; MW 500; Wako) in distilled water. An appropriate volume of the mixture (4 µl per spot in a 96-well plate or 22 µl per spot in a 24-well plate) was spotted in each well of a microtiter plate. When all the wells had been similarly treated, the plate was dried in a vacuum chamber for 1 h. To investigate transfection efficiencies and gene-knockdown efficiencies of siRNAs, we placed cells (0.4–2 ×105 cell per well in a 24-well format) in an appropriate medium (DMEM with 10% FBS or recommended medium by supplier) in each well for reverse transfection. In the case of a conventional transfection method, siRNA was transfected into cells using Lipofectamine 2000 in accordance the supplied protocol. After 24 h, we obtained phase-contrast and fluorescence images of cells in each well with a fluorescence microscope (IX81; Olympus, Tokyo). For quantitative polymerase chain reaction (QPCR) analysis, we extracted mRNAs from cells in each well using a TurboCapture mRNA kit (Qiagen) and performed QPCR using a One Step SYBR PrimeScript RTPCR kit (Takara Bio, Shiga) and an ABI PRISM 7700 system (Applied Biosystems, Foster City, CA, USA) in accordance with the instructions of the manufacturers. The relative efficiency of the
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knockdown of the expression of the gene for CDK2 by anti-CDK2 siRNA was calculated against the control with a non target siRNA. Cytotoxicity induced by reverse transfection from a solid phase was confirmed by staining of dead cells with EthD-1 (Invitrogen). After 24 h of reverse transfection of each cell strain with siRNA, 5 µM EthD-1 in DMEM was added to cells and the cells were incubated for 30 min. Treated cells were washed twice with PBS and after 10 min, dead cells were detected on the basis of the fluorescence of EthD-1. As a control experiment, dead cells were prepared by adding 70% MeOH before staining with EthD-1 (Fig. 2). To investigate the stability of the siRNA mixture in wells of microtiter plates, individual plates, after drying in the vacuum chamber as described above, were vacuum-packed with a desiccant and stored at 4°C.
RESULTS AND DISCUSSION We first investigated the transfection efficiency of our reverse transfection method using siRNA and the toxicity to various cell lines as induced by the method. Figure 1 shows the results for three cell lines (HeLa, SK-BR-3 and MCF7), which were transfected with Cy3-labeled non target siRNA. The transfection efficiency and the localization of siRNA can be assessed from the fluorescent signals. In these cell lines, siRNA was introduced at a high efficiency and was localized in the cytoplasm, which is a prerequisite for the knockdown of mRNA because siRNA generally acts in the cytoplasm. Moreover, cytotoxicity induced by reverse transfection from the solid phase was confirmed by staining dead cells with EthD-1 (Fig. 2). The number of dead cells transfected with siRNA was equal to that not transfected. The number of dead cells transfected with siRNA was markedly smaller than that treated with MeOH. The results show that the toxicity of siRNA transfection to each cell line was very low and no cell death appeared to result from the transfection procedure. Next, we investigated the uniformity of transfection. When cell arrays are used as, for example, described by Ziauddin
FIG. 1. Photographs of three typical lines of cells transfected with Cy3-labeled non target siRNA. (A) HeLa; (B) SK-BR-3; (C) MCF7; upper images, phase-contrast; center images, fluorescence due to Cy3; lower images, merged phase-contrast and fluorescence images; left side of each rectangle, 4× lens; right side of each rectangle, 20× lens. Cells were transfected with Cy3-labeled non specific siRNA in 96-well plates by the wellbased reverse-transfection method. Images were recorded after incubation for 24 h at 37°C in an atmosphere of 5% CO2 in air.
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FIG. 2. Cytotoxicity to three typical lines of cells as induced by reverse transfection from solid phase. (A) HeLa; (B) SK-BR-3; (C) MCF7; upper images, cells without transfection; center images, cells with transfection of non target siRNA; lower images, MeOH-treated cells without transfection; left side of each rectangle, phase contrast; right side of each rectangle, fluorescence staining with EthD-1. After 24 h of reverse transfection of each cell strain with siRNA, 5 µM EthD-1 in DMEM was added to cells and the cells were incubated for 30 min. The cells were washed twice with PBS and after 10 min dead cells were detected on the basis of the fluorescence of EthD-1. As a control experiment, dead cells were prepared by adding 70% MeOH to cells before staining with EthD-1.
and Sabatini (1) and us (4), the spotted solution of transfection reagents dries up immediately and uniformly, because the volume of the transfection solution is very small (10 nl– 25 nl per spot). However, in our new method, because the transfection mixture used to coat the wells does not dry up immediately, the mixture tends to aggregate on the sides of the wells as a result of surface tension, which inhibits uniform transfection. Therefore, we added polyvinyl alcohol (PVA) to our transfection mixture. PVA, which is utilized generally as a dispersant or emulsifying agent, is very strongly hydrophilic and it lowers the surface tension of aqueous solutions. Therefore, we expected that the addition of PVA would promote the uniform spreading of the transfection mixture throughout each well. We postulated that PVA is not toxic to cells because PVA is a physiologically inactive material that has been approved as a resin for food packaging by the US Food and Drug Administration (FDA). We also added dextran to the transfection mixture to enhance transfection efficiency and the spreading of the transfection solution over the entire surface of each well. It has been reported that a high concentration of sucrose in the transfection mixture contributes to the stability of the transfection reagent complex and increases transfection efficiency (11). However, a high concentration of sucrose interferes with the immobilization of the transfection complex on a solid surface; the complex spreads beyond the area of the spot upon addition of the medium in which cells are suspended and, as a result, even cells outside the area of the spot are transfected (11). Although this is major problem for arraybased reverse transfection, it is an advantage for well-based reverse transfection. We found that various polysaccharides,
FIG. 3. Images of siRNA on the lower right side of a well (A) before and (B) after well-based reverse transfection. Left images show Cy3labeled siRNA in the transfection mixture prior to cell seeding (A). Right images show Cy3-labeled siRNA and cells in the same region of the well, after cell seeding and incubation for 16 h as described in the text (B). Images were obtained at two magnifications, as indicated.
including glucose, sucrose and dextran, enhanced the efficiency of transfection (data not shown). Figure 3 shows fluoresence images of siRNAs on the lower right side of a well before and after well-based reverse transfection. The siRNA was labeled with Cy3 to allow us
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TABLE 1. Gene-knockdown efficiency of anti-CDK2 siRNA after well-based reverse transfection of various lines of cells Knockdown efficiency (%) HeLa Adenocarcinoma Cervix 97 T-47D Ductal carcinoma Mammary gland; breast 94 SK-BR-3 Adenocarcinoma Mammary gland; breast 96 MCF7 Adenocarcinoma Mammary gland; breast 97 MDA-MB-231 Adenocarcinoma Mammary gland; breast 87 MDA-MB-435S Ductal carcinoma Mammary gland; breast 86 MDA-MB-468 Adenocarcinoma Mammary gland; breast 88 HCC1428 Adenocarcinoma Mammary gland; breast 46 HCC1806 Primary acantholytic squamous cell carcinoma Mammary gland; breast 82 HCC1954 Ductal carcinoma Mammary gland; breast 92 AU565 Adenocarcinoma Mammary gland; breast 72 HEK293 – Kidney 97 HepG2 Hepatocellular carcinoma Liver 98 NT2 Malignant pluripotent embryonal carcinoma Testis 94 SHSY5Y Neuroblastoma Brain 85 hMSC – Mesenchymal stem cell 94 The relative efficiency of the knockdown of expression of the gene for CDK2 with anti-CDK2 siRNA was calculated against the control with a non target siRNA by QPCR analysis. Cell line
Source
to visualize siRNA that was attached to the solid phase before transfection and to localize it in the cytoplasm after transfection. Images on the left show Cy3-labeled siRNA in the transfection mixture prior to cell seeding. After cell seeding and incubation for 16 h, we photographed the labeled siRNA and cells in the same region of the well (Fig. 3B). As shown in Fig. 3, before reverse transfection, the attached transfection mixture, including siRNA, did not spread uniformly in each well even though PVA induced some spreading of the transfection mixture (Fig. 3A, upper left). Moreover, Cy3-labeled siRNA aggregated as plaques on the solid phase (Fig. 3A, lower left). However, after reverse transfection, the attached siRNA was present inside cells and the efficiency of transfection was uniform in all regions of the well (Fig. 3B). Conventional liquid-based transfection with liposomes, such as Lipofectamine 2000, requires 30 times the volume of transfection mixture as that used in our method. We cannot assume, therefore, that the transfection mixture becomes resuspended in the medium from the solid surface and then siRNA is introduced into cells. Two mechanisms might be involved: transfection might occur from the interface between cells and the solid mixture by phagocytosis; a density gradient formed by dextran might inhibit diffusion components of the redissolved transfection mixture. Next, we investigated the efficiency of gene knockdown in 15 lines of human cells including hMSCs (Table 1). The relative efficiency of the knockdown of the expression of the gene for CDK2 by anti-CDK2 siRNA was calculated against the control with a non target siRNA by QPCR analysis. Almost all cells were transfected with the specific siRNA (anti-CDK2) at a high efficiency. Only Hcc1428 cells derived from a human adenocarcinoma were resistant to the knockdown of the CDK2 gene (46%). Even hMSCs, which are difficult to transfect and gene knockdown by conventional methods, were transfected and the CDK gene was suppressed at a high efficiency (94%). To compare our well-based method with a conventional method, we used anti-CDK2 siRNA to transfect various cell lines by each method. After incubation
Organ (human)
FIG. 4. Comparison of the gene-knockdown efficiency of specific anti-CDK2 siRNA between well-based reverse transfection and conventional transfection. In the case of respective transfection methods, namely conventional transfection and reverse transfection, siRNA was transfected into cells using Lipofectamine 2000 in accordance the procedures shown in materials and methods. After transfection of indicated cells with anti-CDK2 siRNA by each method and incubation for 24 h, the levels of expression of the gene for CDK2 were compared as described in the text. The relative efficiency of the knockdown of expression of the gene for CDK2 with anti-CDK2 siRNA was calculated against the control with a non target siRNA by QPCR analysis.
for 24 h, we compared the relative level of expression of the gene for CDK2 with that of the control with a non target siRNA in each cell line by QPCR (Fig. 4). In all types of cell examined, gene expression was repressed more strongly by our method than by the conventional method. In particular, the level of expression of the gene for CDK2 was decreased to 3% of the normal level in both HeLa and MCF7 cells. One of the advantages of reverse transfection from the solid phase is the long-term stabilization of the reagent mixture that includes DNA or siRNA on the solid phase (12). Therefore, we examined whether it is possible to use plates that had been precoated with the transfection mixture. To
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ACKNOWLEDGMENTS This study was performed as part of “The Project for Development of Analysis Technology for Gene Functions with Cell Arrays”, which was entrusted by the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
REFERENCES
FIG. 5. Relative gene-knockdown activity of siRNA after transfection in wells of stored plates. Plates with attached transfection mixture that included control siRNA (anti-CDK2) were stored at 4°C under a vacuum. Then HeLa cells were seeded in wells of stored plates at the indicated times and gene-knockdown efficiency was evaluated in terms of the level of expression of the gene for CDK2 by QPCR analysis.
establish and evaluate a procedure for storing treated plates, we prepared plates with the transfection mixture that included the specific siRNA (anti-CDK2) and stored them at 4°C under a vacuum for 210 d. We seeded HeLa cells in wells of stored plates at intervals and evaluated the geneknockdown efficiency of the specific siRNA in terms of the expression of the gene for CDK2. Figure 5 shows the relative gene-knockdown activity of siRNA on stored plates over time. We did not detect any reduction in gene-knockdown efficiency for 210 d. Thus, the transfection mixture was extremely stable and the treated plates may be stored for a long time under easily controlled conditions. Our new system provides an inexpensive and stable tool for the high-throughput analysis of gene knockdown. Although array-based reverse transfection has advantages in terms of miniaturization, our efficient and inexpensive method has the advantage of enabling the inclusion of various soluble factors, such as humoral factors, drugs and ligands, that affect gene expression, because the liquid phase is partitioned within the individual wells of each microtiter plate.
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