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Transfection Starting Points for Mammalian Cells
As with other small nucleic acids, such as siRNAs and antisense oligonucleotides, the efficiency with which mammalian cells are transfected with miRNA mimics will vary according to cell type and the transfection agent used. The optimal concentration used for transfections should be determined empirically.
General Transfection Starting Points for microRNA mimics in Cultured Mammalian Cells
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Plate Format
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96 wells
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24 wells
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12 wells
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6 wells
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Transfection Reagents
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0.3–1.0 μL
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1–3 μL
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2–4 μL
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3–6 μL
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miRNA Mimics b
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3 pmol
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15 pmol
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30 pmol
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75 pmol
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Cell Density c
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6,000 cells/well
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40,000 cells/well
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80,000 cells/well
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200,000 cells/well
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Final Volume per Well
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0.1 mL
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0.5 mL
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1.0 mL
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2.5 mL
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a. Refer to the instructions provided with your transfection reagent for the recommended volume.
b. The amount shown results in a final miRNA mimics concentration of 30 nM. The amount of miRNA mimics required for maximal miRNA mimics activity will vary among cell types. For a 96-well plate and 100 μL final transfection volume, 3 pmol of a 5 μM oligonucleotide solution is 0.6 μL. Robotic pipettors may require volumes of 2–5 μL for accurate pipetting. To increase pipetting volumes and accuracy when preparing transfection complexes, we recommend first making a plate with a dilution of your stock oligonucleotide
c. Optimal cell density will vary among cell types, depending on cell size and growth characteristics. In general, we recommend 30–70% confluency.
Transfection Optimization
Optimizing transfection efficiency is crucial for maximizing miRNA mimics activity while minimizing cytotoxicity. Optimal transfection efficiencies are achieved by identifying an effective transfection reagent for each cell type and by adjusting (in order of importance):
● Amount of transfection reagent
● Amount and type of RNA oligonucleotide
● Cell density at the time of transfection
● Order of transfection (pre-plating cells or plating cells/transfecting in tandem)
● Length of exposure of cells to transfection reagent/microRNA mimics complexes
Most protocols recommend maintaining mammalian cells in the medium used for transfection; this avoids dilution or removal of RNA oligonucleotides from the cells by adding medium or washing the cells with new medium too soon after transfection. We have found that cells typically exhibit greater viability when existing medium is replaced with fresh medium 24 hours after transfection. Replacing medium after 24 hours generally does not change the activity of the transfected miRNA mimics. Once the conditions for optimal transfection efficiency are determined, they should be kept constant from experiment to experiment for a given cell type.
Quality Control
Analytical HPLC of a sample of each purified single-stranded RNA oligonucleotide is used to confirm ≥97% purity.
The annealed miRNA mimics are analyzed by nondenaturing gel electrophoresis.
References
1. Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433(7027):769–773. Epub 2005 Jan 30.
2. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843–854.
3. Wightman B, Ha I, Ruvkun G (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75(5):855–862.
4. Yekta S, Shih IH, Bartel DP. 2004. MicroRNA-directed cleavage of HOXB8 mRNA. Science
304(5670):594–596.
5. Bagga S, Bracht J, Hunter S, Massirer K, Holtz J, Eachus R, Pasquinelli AE (2005) Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122(4):553–563.
6. Jing Q, Huang S, Guth S, Zarubin T, Motoyama A, Chen J, Di Padova F, Lin SC, Gram H, Han J (2005) Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120(5):623–634.
7. Schramke V, Sheedy DM, Denli AM, Bonila C, Ekwall K, Hannon GJ, Allshire RC. 2005.
RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription. Nature 435(7046):1275–1279.
8. Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol. 29(1):23–39.
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