Bioluminescence images of three mice after intravenous firefly luciferase mRNA delivery, each showing a bright signal concentrated over the liver. Region-of-interest values are 6.205e+06, 7.032e+06 and 3.660e+06. — In vivo transfection of firefly luciferase mRNA, injected IV at 0.2 mg/kg using the reagent supplied by this kit. ROI 1 6.205e+06, ROI 2 7.032e+06, ROI 3 3.660e+06; scale min 420, max 8113.

AccuLNP™ In Vivo Transfection Kit

Catalog#: NTP-0400
Size
Quantity
Price (USD): $189

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mRNA
siRNA

Product Overview

AccuLNP is a cell transfection reagent mediated by lipid nanoparticles (LNPs) and can be used for the transfection of various types of nucleic acids, such as siRNA, mRNA, and CRISPR mRNA/sgRNA. This kit is specifically designed for in vivo animal studies. AccuLNP efficiently combines with nucleic acid molecules to form lipid–nucleic acid complexes and effectively releases nucleic acid molecules within cells to achieve their biological functions. This reagent is characterised by high transfection efficiency, low cytotoxicity, and ease of use.

Product Detail

Catalog#	Name	Small	Medium	Large
NTP-0400-L	Reagent L (liquid)	50 µL	120 µL	240 µL
NTP-0400-A	Reagent A (liquid)	80 µL	200 µL	400 µL
NTP-0400-ST	Reagent ST (liquid)	25 mL	25 mL	25 × 2 mL
N/A	Amicon® Ultra centrifugal filters (100 kDa)	2	2	2
N/A	1.5 mL sterile microtubes	4	4	4

Storage condition: -20 °C
Shelf life: 12 months
Encapsulation time: ~60 s
Format: Three-reagent liquid kit with centrifugal filters and sterile microtubes

Protocol

Table 1. Reagent use in the preparation of transfection complex.

For one well	Reagent A volume	RNA volume (at 1 µg/µL)	Reagent L volume	Reagent ST volume	RNA concentration in transfection complex at step (5)	Volume of transfection complex at step (5)
Per preparation	19.25 µL	22 µL	13.75 µL	1045 µL	20 µg/mL	1100 µL

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 an RNA concentration of 20 µg/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 Reagent A and Reagent ST at 37 °C. Apply vortex mixing to ensure homogeneous solutions are obtained. Cool to room temperature before use.
2. Thaw Reagent L at 37 °C. The solubilisation time is at least 10 minutes.
  
  Note: Apply vortex mixing and invert the vial multiple times during solubilisation. Visually check and ensure all precipitates are completely dissolved. Cool to room temperature before use.
3. Follow aseptic techniques for the preparation of transfection complex at room temperature. In a new sterile centrifuge tube (supplied in the kit), add 19.25 µL Reagent A and 22 µg (1 µg/µL) of firefly luciferase mRNA to make the final volume 41.25 µL. Use a pipette to mix evenly.
4. Add 13.75 µL of the Reagent L solution 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 55 µL.
5. Add 1045 µL of the Reagent ST solution into the transfection complex prepared in step (4) and shake the centrifuge tube gently until mixed evenly. The total sample volume at this step is 1100 µL.
6. The resulting RNA–LNP can be purified using the supplied Amicon® Ultra centrifugal filters. Transfer the sample to a centrifugal filter. Draw a mark on the outer wall of the filter 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 100 kDa MWCO centrifugal filters; alternatively, the user may use one centrifugal filter with a longer centrifuge time.
7. Centrifuge the sample for 5 minutes at 2000 relative centrifugal force (RCF) to concentrate the sample to about 250–500 µL. To maintain the stability of the RNA, it is recommended the centrifugation is performed at 4 °C.
8. Dilute the sample using Reagent ST to the mark drawn in step (6), then use a pipette to mix evenly. Repeat step (7). Do this step at least twice for purification.
9. To collect the sample, dilute the sample with Reagent ST to the mark drawn in step (6), then use a pipette 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 1100 µL transfection solution with an RNA concentration of 20 µg/mL has been prepared.
10. The above prepared transfection complex can be directly used for animal work. Do not use a 0.22 µm membrane to filter.
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 a 0.22 µm membrane to filter the RNA–LNP mix. The injection volume will vary based on the experimental design and final RNA concentration. As an example, to dose a 25 g mouse at 0.2 mg/kg, an injection volume of 250 µL of RNA–LNP at 20 µg/mL RNA, made using these instructions, would be needed. The kit user should verify the volume needed for their experiment prior to using the RNA–LNP for any in vivo injections, using the formula below.

Volume of RNA–LNP per dose (µL) = Animal weight (g) × Dosage (µg/g) ÷ RNA–LNP concentration (µg/µL)

Worked example from the protocol above — a 25 g mouse at 0.2 mg/kg needs 250 µL of RNA–LNP at 20 µg/mL.

Notes

Use a 37 °C water bath to accelerate the dissolution of Reagents A, L and ST. Unused reagent can be stored at −20 °C.
The kit supplies a total of two Amicon® Ultra centrifugal filters. If more are needed, the user can order additional Amicon Ultra-4 centrifugal filters, 100 kDa MWCO (Millipore). Note that the supplied Amicon Ultra centrifugal filter is a single-use, disposable device.

Ordering

Catalog number NTP-0400. Net 30 on an institutional purchase order. Prices exclude shipping and applicable tax. For scale-up, custom formats, or bulk pricing, email contact@genveyor.com.