Reagents List

Product Name	Cat.NO.
Hieff Trans™ Booster DNA/RNA Transfection Reagent	40801ES

mNSCs-mRNA Transfection Protocol

Cell Culture and Passaging (T25 Flask)

• Washing: Aspirate old medium and gently rinse with 3–4 mL of room-temperature PBS for 10–20 seconds, then aspirate.  
• Digestion: Add 1 mL trypsin to cover the bottom of the flask and incubate at 37°C for digestion.  
• Detachment: When cells begin to retract and round up, gently pipette the trypsin solution to dislodge the cells.  
• Termination: Add 2–3 mL complete culture medium to stop digestion, and pipette to create a uniform cell suspension.  
• Subculturing: Transfer the suspension to a centrifuge tube, centrifuge at 200 × g for 3–5 minutes, discard supernatant, resuspend pellet in fresh medium, seed into new flasks, and add complete medium for continued culture.

Cell Transfection (96-well-plate)

I. Preparation

1. Cell Preparation:

•  24 hours before transfection, harvest cells using trypsin, resuspend, and count.
• Seed cells into 96-well-plate (or other culture vessels) at an appropriate density with 0.1 mL complete medium per dish.
• Target 60–80% confluency at the time of transfection.

2. Reagent Preparation:

• mRNA: Prepare high-purity mRNA solution.
• Transfection Reagent: Allow Hieff Trans™Booster Transfection Reagent to warm to room temperature. Gently mix before use.  
• Serum-Free Medium: Prepare serum- and antibiotic-free basal medium (e.g., Opti-MEM) for diluting mRNA and transfection reagent.

II. Complex Formation

1. Dilute mRNA: In a sterile microcentrifuge tube, add 5 μL serum-free medium followed by 20 ng mRNA. Mix gently.

2. Dilute Transfection Reagent: In another sterile tube, add 5 μL serum-free medium and 0.1 μL Hieff Trans™Booster Transfection Reagent. Mix gently.  

3. Combine and Incubate: Add the diluted mRNA solution to the diluted transfection reagent tube. Mix gently by pipetting. Incubate at room temperature for 10–15 minutes to form mRNA-transfection reagent complexes. 

III. Transfection

1. Medium Change: Before transfection, carefully aspirate the old medium and replace with 0.1 mL pre-warmed complete medium.  

2. Add Complexes: Add the incubated mRNA-transfection reagent complexes (~10 μL total) dropwise and evenly to the cells. Gently rock the plate to distribute. 

3. Incubation: Return cells to a 37°C, 5% CO₂ incubator for culture.

IV. Post-Transfection Handling

1. Medium Replacement: 4–6 hours post-transfection, check cell morphology. If significant toxicity is observed, aspirate the medium containing complexes and replace with 0.1 mL fresh pre-warmed complete medium. If cells appear healthy, medium change is optional.  

2. Analysis: After 24–72 hours, assess transfection efficiency or functional outcomes based on experimental design (e.g., fluorescence observation, mRNA or protein detection).

Tips:

1. If significant cytotoxicity occurs, replace medium after 6 h or reduce Enhancer volume by half to mitigate toxicity.  

2. If transfection efficiency is low, increasing the amount of transfection reagent may improve results.  

3. For optimal performance, dilute mRNA and transfection reagent in Opti-MEM rather than DMEM.  

4. The transfection system is compatible with serum and antibiotics; however, mRNA and reagent dilutions must be performed in serum- and antibiotic-free medium.

Experimental Results Analysis

mRNA was transfected into primary mouse neural stem cells(mNSCs), and protein expression, cell morphology, and viability were assessed by immunofluorescence staining.

A*8008                               Hieff Trans™ Booster 

Figure 1. mRNA transfection in primary mouse neural stem cells(mNSCs) using Yeasen Booster DNA/RNA Transfection Reagent versus A*8008. The result demonstrates superior primary cell transfection efficiency of Booster.

Different Cell Culture Vessel Transfection Volumes (for reference only):

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