For ”selecting,” ”removing,” and ”detecting” iPS cells/iPSC-derived cardiomyocytes

[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
Principle
Products List
Example1: Selection (P-0004 CM purifier RNA Switch™)
Example2: Removal of iPS cells (P-0006 iPSC eliminator RNA Switch™)
Example 3: Detection of various cells　(P-0002, P-0003)
Q&A
Product List
Related Product List
Related Information

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
↓
Suicide gene is expressed (switch-on)
↓
Apoptosis (cell death) is induced

Wanted cells

Target miRNA binds within cells
↓
Suicide gene expression is inhibited (switch-off)
↓
RNA Switch™ (mRNA) is degraded in approximately 24 hours

Products List

Target cells	Purpose	Required product		Function	Complementary sequence of miRNA	Reporter gene	Result
Target cells	Purpose	Manufacturer code	Product Name	Function	Complementary sequence of miRNA	Reporter gene	Result
Myocardial/iPS cells	To check the transfection efficiency	P-0001	Control detector RNA Switch™	To determine the transfection efficiency	None	Fluorescent protein gene	The transfection efficiency is checked after transfection. It is desirable to achieve a transfection efficiency of at least 30%, ideally 50% to 60%. The required measures include proficiency at transfection procedure and changing transfection reagents.
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.
Cardiomyocytes	To detect	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
iPS cells	To detect	P-0003	iPSC detector RNA Switch™	To induce non-iPS cells to fluorescent	miR-302	Fluorescent protein gene	The same as above
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	Puromycin kills all cells not transfected with P-0007.	–	–
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.

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

Day 0	Seeding: 6-well plate, 8.0-10.0 x 10⁵ cells/well ↓ Incubation: 37℃ and 5% CO₂ (24 hours)
Day 1	Medium exchange ↓ Incubation: 37℃ and 5% CO₂ (1-2 hours) ↓ Reagent preparation, transfection** ↓ Incubation: 37℃ and 5% CO₂ (4 hours) ↓ Medium exchange (containing 2 µg/mL puromycin) ↓ Incubation: 37℃ and 5% CO₂ (24 hours)

Day 2 Medium exchange (containing 2 µg/mL puromycin)
↓
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.

Day 2	Medium exchange (containing 2 µg/mL puromycin) ↓ Incubation: 37℃ and 5% CO₂ (24 hours)
Day 3	Analysis: Microscope or c-TnT FACS

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™

[aceRNA Technologies] 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.

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