AccuLNP Cell Transfection Kit (For in vivo applications) NTP-0103

Product Introduction 

AccuLNP is a cell transfection reagent mediated by lipid nanoparticles (LNP) and can be used for the transfection of different types of nucleic acids, for example siRNA, mRNA, CRISPR mRNA/sgRNA and other RNA-based biological molecules. This kit is specifically designed for in vivo animal studies. AccuLNP can efficiently combine with nucleic acid molecules to form (lipid molecules + nucleic acid) complexes and can effectively release nucleic acid molecules within cells to achieve their biological functions. This reagent has the characteristics of high transfection efficiency, low cytotoxicity, and easy operation. 

Storage condition: -20°C 

Shelf life: 12 months 

The kit is good for 24 mL LNP with 20μg/ml RNA. 

Procedures 

The following example is for transfecting Firefly Luciferase mRNA in vivo (mice). The transfection reagent prepared in the example can be used for injection at 50μL at each dosing for at least 20 times at an RNA concentration of 20μg/mL. This protocol can be proportionally scaled up or down as needed. 

Prepare the lipid/RNA complex for transfection. 

(1) Take the kit out of the freezer and thaw Reagents A and E at room temperature or in a 37°C water bath. 

(2) Add 300μL of Reagent E to Reagent L, use a 37°C water bath to accelerate solubilization, and the solubilization time is at least 20 minutes. 

(3) In a new sterile centrifuge tube (supplied in the kit), add 19.25μL Reagent A and 22μg (1μg/μL) of Firefly Luciferase mRNA to make the final volume as 41.25μL. Use a pipette to mix evenly. For other types of RNA, users can adjust the amount of RNA and test the transfection efficiency to obtain the optimal ratio of lipid to nucleic acid. 

(4) Add 13.75μL of the solution prepared in step (2) to the RNA solution prepared in step (3) and use a vortex mixer to mix quickly for at least 15 seconds. The resulting total volume is 55μL. 

(5) Take a tube of Reagent ST, add 14 mL of sterile pure water, and mix thoroughly until dissolved. 

(6) Add 1045μL of the solution prepared in step (5) into the lipid /nucleic acid mixture prepared in step (4) and shake the centrifuge tube gently until mixed evenly. 

(7) The resulting RNA-LNP can be purified by using the supplied Amicon® Ultra Centrifugal Filters. Transfer the sample to a centrifugal filter. Draw a mark on the outer wall of the filter wall to indicate the volume of the sample. If the user has scaled up the protocol to accommodate more volume, it is recommended to use additional 100kDa MWCO centrifugal filters, alternatively, the user may use one centrifugal filter with a longer centrifuge time. 

(8) Centrifuge the sample for 5 minutes at 3000 relative centrifugal force (RCF) to concentrate the sample to about 350-500μl. To maintain the stability of the RNA, it is recommended the centrifuge is performed at 4°C. 

(9) Dilute the sample using Reagent ST to the mark drawn in step (7), then use a pipette to mix evenly. Repeat step (8). Do this step at least twice for purification. 

(10) To collect the sample, dilute the sample with Reagent ST to the mark drawn in step (7), then use a pipet to mix evenly and dislodge any material retained on the filter membranes. Carefully transfer all the sample volume to a new sterile centrifuge tube (supplied in the kit). At this point, a total of 1100μL transfection solution with the RNA concentration of 20μg/ml has been prepared. 

In vivo transfection 

The above prepared RNA-LNP transfection mix can then be used directly for in vivo injections. Please note all reagents are supplied for in vivo use and please do not use 0.22μm membrane to filter the RNA-LNP mix. 

Notes 

1. Use a 37°C water bath to accelerate the dissolution of Reagent L in Reagent E. The dissolution time is at least 20 minutes. Unused solution of Reagent L in Reagent E can be stored at -20°C. Thaw and dissolve again in a 37°C water bath for no less than 20 minutes before next use. 
2. During the preparation of transfection reagents, to avoid liquid loss, please use a centrifuge (e.g., Mini Centrifuge) to collect the liquid at the bottom of the centrifuge tube after mixing the reagents in the tube. 
3. For different types of RNA, users can adjust the amount of RNA and test the transfection efficiency to obtain the optimal ratio of lipid to nucleic acid. 
4. Unused reagent ST can be stored at -20°C. Thaw and dissolve in room temperature or 37°C water bath before next use.