[aceRNA Technologies] RNA Switch™
This series of products comprises artificial mRNA produced using "RNA Switch™" technology invented by Professor Hirohide Saito of Center for iPS Cell Research and Application, Kyoto University and colleagues, and can be used to identify or select only specific cell types.
RNA Switch™, which uses a suicide gene, can be used to select cells quickly and easily without special machinery when transfected into cells.
Features
- Requires no large equipment
- Requires no antibody
- Less harmful to cells
- Can be used to process large numbers of cells
Principle
RNA Switch™ = Artificially synthesized "mRNA"
RNA Switch™, an artificial mRNA, contains the "complementary sequence of target miRNA" and the "fluorescent protein gene (for detection) or suicide gene (for selection and removal)." When the target miRNA is present in cells transfected with RNA Switch™, the mRNA (RNA Switch™) is degraded. In the absence of the target miRNA, on the other hand, the gene contained in RNA Switch™ is expressed.
For selection (see left panel)
Unwanted cells
Target miRNA is not present in cells
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Suicide gene is expressed (switch-on)
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Apoptosis (cell death) is induced
Wanted cells
Target miRNA binds within cells
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Suicide gene expression is inhibited (switch-off)
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RNA Switch™ (mRNA) is degraded in approximately 24 hours
Products List
| Target cells | Purpose | Required product | Function | Complementary sequence of miRNA | Reporter gene | Result | |
|---|---|---|---|---|---|---|---|
| Manufacturer code | Product Name | ||||||
| Myocardial/iPS cells | To check the transfection efficiency | P-0001 | Control detector RNA Switch™ | To determine the transfection efficiency | None | Fluorescent protein gene |
|
| Cardiomyocytes | To detect | P-0001 | Control detector RNA Switch™ | Control (to induce all cells to fluorescent) | None | Fluorescent protein gene | Comparison with a control shows the extent to which unwanted cells are present. |
| P-0002 | CM detector RNA Switch™ | To induce non-Cardiomyocytes to fluorescent | miR-1 | Fluorescent protein gene | |||
| iPS cells | To detect | P-0001 | Control detector RNA Switch™ | Control (to induce all cells to fluorescent) | None | Fluorescent protein gene | The same as above |
| P-0003 | iPSC detector RNA Switch™ | To induce non-iPS cells to fluorescent | miR-302 | Fluorescent protein gene | |||
| Cardiomyocytes | To select (kill non-myocardial cells) |
P-0004 | CM purifier RNA Switch™ | To kill non-myocardial cells | miR-1 (Cardiomyocytes specific) |
Suicide gene | Surviving cells 1. Cardiomyocytes transfected with P-0004 and P-0007 2. Cells transfected with P-0007 but not with P-0004 (very few) |
| P-0007 | puro resistant mRNA™ | Puromycin kills all cells not transfected with P-0007. | None | puromycin-resistant gene | |||
| - | puromycin | - | - | ||||
| iPS cells | To remove (kill iPSCs) |
P-0006 | iPSC eliminator RNA Switch™ | To kill iPS cells (inhibits the expression of the P-0007-derived, puromycin-resistant gene) | miR-302 (iPSC specific) |
puromycin-resistant gene | Only iPS cells are removed. |
Example1: Selection (P-0004 CM purifier RNA Switch™)
Co-transfection of CM purifier RNA Switch™ (P-0004) and puro resistant mRNA™ (P-0007) into a cell population containing human cardiomyocytes allows large amounts of human cardiomyocytes to be more highly purified at a lower cost than conventional methods using a cell sorter/antibody.
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Cell Sorter
RNA Switch™
Requires expensive equipment → Requires no special device Depends on the presence or absence and function of the antibody → Requires no antibody Damages cells (flow rate, low pH) → Less damage to cells Treats only small numbers of cells → Can treat large numbers of cells
Has resolved problems with cell sorter
Application Data
Protocol overview
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Day 0 Seeding: 6-well plate, 8.0-10.0 x 105 cells/well
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Incubation: 37℃ and 5% CO2 (24 hours)Day 1 Medium exchange
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Incubation: 37℃ and 5% CO2 (1-2 hours)
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Reagent preparation, transfection**
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Incubation: 37℃ and 5% CO2 (4 hours)
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Medium exchange (containing 2 µg/mL puromycin)
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Incubation: 37℃ and 5% CO2 (24 hours) -
Day 2 Medium exchange (containing 2 µg/mL puromycin)
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Incubation: 37℃ and 5% CO2 (24 hours)Day 3 Analysis: Microscope or c-TnT FACS ** Co-transfection of CM purifier RNA Switch™ (P-0004) and puro resistant mRNA™ (P-0007) results in higher selectivity.
Example2: Removal of iPS cells (P-0006 iPSC eliminator RNA Switch™)
iPSC eliminator RNA Switch™ (P-0006) can be used to eliminate human iPS cells alone, by specifically controlling the expression of the puromycin-resistant gene according to the activity of miR-302, which is specifically active in human pluripotent stem cells.
Application Data: Comparison before and after removal of iPS cells
P-0006 iPSC eliminator RNA Switch™
Example 3: Detection of various cells (P-0002, P-0003)
CM detector RNA Switch™ (P-0002) and iPSC detector RNA Switch™ (P-0003) can be used to detect unwanted cells other than cardiomyocytes and human iPS cells, respectively, using fluorescence. Use of these products is recommended in order to evaluate the cell transfection efficiency of RNA Switch™. Please use Control detector RNA Switch™ (P-0001) together as a control.
Q&A
How many times can a single instance of the product be used?
It requires three 6-well plates. The quantity of this product is 9 µg/tube, and 0.5 µg/well RNA Switch™ is used when cells are seeded in a 6-well plate at a density of 5 x 105 cells/well.
How long does RNA Switch™ remain in cells after transfection?
It disappears in 24 hours.
Can RNA Switch™ be used for electroporation transfection?
Yes, it can.
Product List
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For research use or further manufacturing use only. Not for use in diagnostic procedures.
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