A Highly Efficient siRNA Transfection Method in Primary Cultured Cortical Neurons

Materials and reagents

Biological materials

1. C57BL/6L mouse [Ji’nan Pengyue Laboratory Animal Breeding Co., Ltd. (China), origin: Ji’nan Shandong, China]

Reagents

1. Poly-D-lysine (Sigma, catalog number: P6407-10X5MG)

2. Neurobasal^TM medium (1×) (Gibco, catalog number: 21103049)

3. B-27 supplement (50×) (Gibco, catalog number: 17504044)

4. siRNA transfection reagent (Kermey, catalog number: MLR0201)

5. Lipofectamine^TM RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number: 13778150)

6. GlutaMAX^TM (100×) (Gibco, catalog number: 35050061)

7. Dulbecco’s modified Eagle medium (DMEM) basic (1×) (Gibco, catalog number: 11995065)

8. Penicillin-streptomycin liquid (100×) (Solarbio, catalog number: P1400)

9. Phosphate-buffered solution (PBS), 0.01 M (powder, pH 7.2–7.4) (Solarbio, catalog number: P1010)

10. Opti-MEM (1×) (Gibco, catalog number: 31985047)

11. Fluorescent dye-labeled siRNA negative control (GenePharma)

12. Standard negative control (siNC) (GenePharma, sequence: UUCUCCGAACGUGUCACGUtt)

13. Custom siRNA targeting PHGDH (GenePharma, sequence: UCGGCAGAAUUGGAAGAGAtt)

14. Triton X-100 (Solarbio, catalog number: T8200)

15. Albumin (bovine fraction V) (BSA) (MeilunBio, CAS: 9048-46-8)

16. DAPI (Solarbio, catalog number: S2110)

17. Paraformaldehyde (PFA), 4% (Solarbio, catalog number: P1110)

18. Primary antibody used in this study: PHGDH (ProteinTech, catalog number: 14719-1-AP)

19. Second antibody used in this study: HRP-conjugated Affinipure goat anti-rabbit IgG (H+L) (SA00001-2) (ProteinTech)

20. CCK8 Assay kit (MeilunBio, CAS: MA0218)

21. Fetal bovine serum (FBS) (Sigma, catalog number: F0193-500ML)

Solutions

1. Triton X-100, 0.5% (see Recipes)

2. BSA, 5% (see Recipes)

3. Neurobasal medium (see Recipes)

4. Trypsin, 0.025% (see Recipes)

5. Complete medium (see Recipes)

6. PDL 10× (see Recipes)

7. PDL 1× (see Recipes)

Recipes

1. Triton X-100, 0.5%

2. BSA, 5%

3. Neurobasal medium

4. Trypsin, 0.025%

5. Complete medium

6. PDL 10×

7. PDL 1×

Laboratory supplies

1. 24-well tissue culture plate (JET, catalog number: TCP011124)

2. 24-well round coverslip (WHB, catalog number: WHB-24-CS)

3. 50 mL centrifuge tube (LABSELECT, catalog number: CT-012-50)

4. 15 mL centrifuge tube (LABSELECT, catalog number: CT-002-15A)

5. 1.5 mL centrifuge tube (LABSELECT, catalog number: MCT-001-150)

6. 0.22 μm PES syringe filter (Biosharp, catalog number: BS-PES-22)

7. 50 mL syringe (Beyotime, catalog number: FS850-20pcs)

8. 200 μL RNase-free pipette (Biosharp, catalog number: BS-200-TRS)

9. 10 μL RNase-free pipette (Biosharp, catalog number: BS-RT-10)

Equipment

1. Dissection microscope (RWD, model: 77001S)

2. Dissection tools: forceps (RWD, model: FC4001R-11) and iris scissors (RWD, model: S12003-09)

3. Biosafety cabinet (Esco, model: AC2-4S1)

4. CO₂ incubator (Thermo Scientific, model: Forma Steri-Cycle i160 CR)

5. SpectraMax ABS (Molecular Devices, USA)

6. Low-speed desktop centrifuge (BAIOU, model: TDL-320)

7. Amersham ImageQuant^TM 800 GxP (Cytiva, model: 29653452)

8. Confocal microscope (Nikon, model: Nikon C2)

Software and datasets

1. Image J [NIH, Java 1.8.0_345 (64-bit)]

Procedure

A. Preparation of cortical neuron cultures

1. One day in advance, coat the required well plates or culture dishes with 1× PDL, ensuring the bottom of each well is completely covered. The following day, aspirate the PDL and wash the wells three times with sterile PBS. Allow the plates to dry in an incubator before use.

2. Euthanize a pregnant C57BL/6 mouse at embryonic day 15–18 (E15–E18) using CO₂ asphyxiation. Open the abdominal cavity, carefully remove the fetuses, and immediately transfer them into ice-cold PBS supplemented with 1% penicillin-streptomycin.

3. Carefully remove the fetal membranes with fine scissors and separate the heads at the neck region. Under a dissecting microscope, peel away the skin from the heads, then open the skulls along the midline to expose the brains. Gently remove the meninges, isolate the cortical tissue, and transfer it into pre-chilled DMEM. Perform all procedures on ice to maintain tissue viability.

4. Aspirate the DMEM and digest the tissue with 0.025% trypsin-EDTA. Gently triturate the tissue using a pipette to break it into smaller fragments. Incubate at 37 °C for 15–20 min, gently agitating the mixture three times during incubation to ensure uniform digestion.

5. Terminate the digestion by adding complete medium. Filter the cell suspension once, then centrifuge at 322× g for 5 min.

6. After centrifugation, carefully discard the supernatant and resuspend the pellet in fresh, prewarmed neurobasal medium. Filter the resuspended cell suspension again through a filter mesh. For cell counting, load 10 μL of the filtered suspension into a clean hemocytometer. Count the cells under a microscope, calculate the average count, and multiply this value by the total volume of the cell suspension in milliliters to determine the total cell number.

7. Seed the cells according to the experimental requirements (in this protocol, plate 1 × 10⁶ cells per well in a 12-well plate).

8. Replace half of the medium with fresh, prewarmed neurobasal medium every 3 days and routinely monitor the neuronal health and morphology.

B. Primary neuron transfection with siRNA transfection reagent from Kermey

1. Perform transfection when neurons reach 4–5 days in vitro (DIV4–5), ensuring a minimum culture period of 3 days prior to transfection. This protocol is designed for a 24-well plate format (refer to Table 1 for scaling adjustments to other plate formats). Prepare transfection complexes using a 20 μM stock solution of siRNA.

Note: Table 1 specifies reagent volumes for different culture plate formats.

Table 1. Transfection complex preparation

2. To prepare the siRNA-transfection complex for a single well of a 24-well plate, begin by adding 7 μL of buffer A into a sterile microcentrifuge tube. Next, add 0.5 μL of siRNA solution (from the 20 μM stock) and mix thoroughly by vortexing or pipetting. Then, add 2 μL of siRNA transfection reagent and mix completely. Incubate the mixture at room temperature for 10 min. Subsequently, add 45 μL of Opti-MEM medium and mix by pipetting up and down approximately five times to yield the final transfection complex, which is now ready for immediate use (Figure 1).

3. For transfection, gently add the prepared complex dropwise to the neuronal culture in the 24-well plate. Gently rock the plate back and forth 3–5 times to ensure uniform distribution of the solution.

4. After transfection, incubate the cells at 37 °C in a 5% CO₂ atmosphere. For optimal assessment of knockdown efficiency, analyze protein levels 48–72 h post-transfection. In this protocol, neuronal protein samples were collected at a 48-h time point for subsequent analysis.

Figure 1. Steps of siRNA transfection

C. Primary neuron transfection with Lipofectamine^TM RNAiMAX transfection reagent (Thermo Fisher Scientific)

1. Perform transfection when neurons have been in culture for 4–5 days in vitro (DIV4–5).

2. Dilute Lipofectamine^® RNAiMAX reagent in Opti-MEM medium (Table 2).

3. Dilute siRNA-standard negative control (siNC) or siPHGDH in Opti-MEM.

4. Combine the diluted siRNA with the diluted Lipofectamine^® RNAiMAX reagent at a 1:1 ratio.

5. Incubate the mixture for 5 min at room temperature.

6. Add the siRNA–lipid complex dropwise to the neuronal culture.

7. After 6–8 h, replace the medium containing the transfection mixture with prewarmed neurobasal medium.

Note: Table 2 specifies reagent volumes for different culture plate formats.

Table 2. Transfection complex preparation of Lipofectamine^TM RNAiMAX transfection reagent

D. Analyze neuronal viability and transfection efficiency

1. Cell viability analysis: Analyze neuronal morphology by microscopy 48 h post-transfection.

Note: Neurons transfected with Lipofectamine^® RNAiMAX reagent exhibited extensive cell death, whereas neurons transfected with the siRNA transfection reagent from Kermey displayed normal morphology. To further assess cell viability, we performed a CCK-8 assay. Primary cortical neurons seeded in 48-well plates were subjected to transfection. After 48 h, CCK-8 reagent was added to each well, followed by incubation for an additional 4–6 h. We measured the absorbance (OD) at 450 nm using a SpectraMax ABS microplate reader to evaluate cell viability. As shown in Figure 2, cell viability in the Lipofectamine RNAiMAX group was reduced to approximately 50% compared to the control group, whereas only a slight decrease was observed with the Kermey siRNA transfection reagent.

Figure 2. Cell viability analysis of neurons after transfection with siNC using Kermey transfection reagent for 48 h. n  =  4. The data are means ± SD, for all panels: *P  <  0.05, **P  <  0.01, ***P  <  0.001, n.s. no significance by one-way ANOVA analysis.

2. siRNA transfection efficiency analysis

a. Forty-eight hours after transfecting neurons with fluorescently labeled siRNA using the Kermey reagent, remove the cell-covered coverslips from the incubator and rinse them three times with PBS (1×), allowing 5 min per wash.

b. Next, fix the samples with 4% PFA for 10 min, followed by three additional 5-min washes in PBS.

c. Permeabilize the cells with 0.5% Triton X-100 for 10 min and wash three times with PBS, each for 5 min.

d. Afterward, block nonspecific binding sites by incubating the samples in 5% BSA for 1 h. Carefully lift the coverslips from the wells using a bent 1 mL syringe needle, and invert them cell-side down onto a glass slide precoated with a drop of DAPI-containing mounting medium. Seal the edges of the coverslip with fingernail polish and allow the slide to dry completely at room temperature for 5–10 min.

e. Finally, image the samples using a Nikon C2 confocal microscope following standard operating protocols.

Note: Most neurons were successfully transfected with negative control siRNA (Figure 3B), as indicated by the evenly distributed green fluorescence throughout the cytoplasm, demonstrating the effective delivery of siRNA using the Kermey transfection reagent.

Figure 3. Morphology of primary cortical neurons and siRNA transfection efficiency. (A) Cortical neurons 48 h after transfection with siNC using Kermey transfection reagent or Lipofectamine^® RNAiMAX reagent. (B) Confocal microscopy images showing cortical neurons transfected with fluorescent dye-labeled siRNA using Kermey transfection reagent. The siRNA is localized in the cytoplasm (green), while nuclei are stained with DAPI. Scale bars represent 10 μm. The data are representative of three independent experiments.

3. Analysis of gene targeting efficiency

To analyze gene knockdown efficiency, primary neurons were transfected with a control siRNA (siNC) and a validated siRNA targeting PHGDH (siPHGDH) [9], as described in section B. Furthermore, 3-phosphoglycerate dehydrogenase (PHGDH) is notable as a key enzyme that functions as the primary rate-limiting enzyme in the serine biosynthesis pathway, facilitating the conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate. After 48 h, we performed the immunoblot analysis. Total proteins were extracted from cells with lysis buffer containing protease (1% P6730) and phosphatase inhibitors (1% P1260). The proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST for 2 h at room temperature, followed by overnight incubation with primary antibodies and a subsequent 1-h incubation with secondary antibodies. Then, membranes were incubated with primary antibodies overnight and secondary antibodies for 1 h. Membranes were exposed to a chemiluminescence detection system. The protein abundance of PHGDH was significantly reduced in neurons transfected with siPHGDH, compared with neurons transfected with siNC (Figure 4).

Figure 4. siRNA transfection reagent from Kermey can effectively knock down the expression of target proteins in neurons. Representative immunoblot analysis of PHGDH expression in primary cultured neurons after transfection with siNC and siPHGDH by using the transfection reagent from Kermey. The data are representative of three independent experiments.

Data analysis

Note: Data are processed using Microsoft Office Excel and analyzed using GraphPad Prism software.

1. Cell viability is determined by measuring the OD at 450 nm. Percent over control was calculated as a measure of cell viability. Calculation is as follows:

Cell survival rate = [(As-Ab)/(Ac-Ab)] × 100%

As: Absorbance of the experimental well (containing cell culture medium, CCK-8, and the test drug).

Ac: Absorbance of the control well (containing cell culture medium, CCK-8, without the test drug).

Ab: Absorbance of the blank well (containing culture medium without cells and the test drug, with CCK-8).

2. We analyzed the western blot results using ImageJ by calculating the ratio of PHGDH band intensity to the corresponding actin band intensity for both the experimental and control groups.

3. One-way ANOVA analysis was used to analyze the difference between the control, lipofectamine, and Kermey groups.

4. All experiments were performed three times unless otherwise indicated.

Validation of protocol

To validate the protocol, we evaluated neuronal morphology, siRNA transfection efficiency, and target gene expression following gene knockdown in neurons. Notably, neurons transfected using Lipofectamine^® RNAiMAX exhibited extensive cell death, whereas those transfected with the siRNA transfection reagent from Kermey maintained normal morphological features (Figure 3A). Furthermore, confocal microscopy revealed efficient transfection, with the majority of neurons successfully delivering the negative control siRNA (Figure 3B). Additionally, western blot analysis confirmed a significant reduction in PHGDH expression in neurons transfected with siPHGDH, compared to those treated with siNC (Figure 4).

General notes and troubleshooting

General notes

1. During the 24–72 h post-transfection period, complete medium replacement is unnecessary when using Kermey transfect reagent. However, partial medium supplementation should be performed as needed to compensate for evaporation, particularly at the edges of the culture vessel.

2. This protocol has been validated exclusively in primary neuron cultures. The transfection efficiency and cellular viability parameters have not been tested in other neural cell types, including but not limited to astrocytes, microglia, oligodendrocytes, or neural progenitor cells.

3. The earliest successful transfection was achieved at DIV3. For earlier timepoints, protocol optimization may be necessary.

4. This procedure is compatible with media heated to 37 °C and subsequently equilibrated to room temperature.

5. When initiating transfections, it is recommended to assess transfection efficiency. Concurrently, select cells that exhibit strong transfection signals and appear morphologically healthy.

6. Use RNase-free pipette tips during transfection to prevent RNA degradation.

Troubleshooting

Problem 1: The siRNA-mediated knockdown effect was not significant.

Possible cause: Variations in cell density and physiological states across cultures might compromise transfection efficacy, as high-density or confluent regions may require higher siRNA concentrations than those specified in our current protocol to achieve effective gene silencing.

Solution: When working with high-density cultures, prepare the siRNA-transfection mixture at an increased concentration (e.g., 1.5× or 2× concentration) while preserving the optimal reagent ratios. However, if cells exhibit poor health prior to transfection, it is strongly recommended to first optimize the cell culture conditions, such as adjusting plating density, refining medium composition, and ensuring strict control of incubation parameters (37 °C, 5% CO₂ with humidity regulation) to improve overall cellular fitness and transfection responsiveness.

Problem 2: Neurons exhibit low viability.

Possible cause: Low cell density may impair siRNA uptake, while excessive siRNA concentrations can induce cytotoxicity, leading to reduced neuronal survival.

Solution: Increase cell number or decrease siRNA concentration.

Acknowledgments

W.X. designed and performed most experiments, analyzed data, and prepared the manuscript; L.Y. helped with neuronal culture; and Y.C. and Z.Z. conceptualized the research, directed the study, and prepared the manuscript. All authors revised the manuscript. This work was supported by the Science and Technology Support Plan for Youth Innovation of Colleges and Universities of Shandong Province of China (2022KJ146), National Natural Science Foundation of China (32470984), and Natural Science Foundation of Shandong Province (ZR2024MH302).

Competing interests

The authors declare no conflicts of interest.

Ethical considerations

Male 9~10 weeks C57BL/6 mice were obtained from Ji’nan Pengyue Laboratory Animal Breeding Co., Ltd. (China). The mice were housed under specific pathogen-free conditions with a constant temperature and humidity on a 12 h light/dark cycle. Food and water were given ad libitum.

All animal experiments were conducted in compliance with National Institutes of Health guidelines and were approved by the institutional animal care and use committee of Qingdao University (QYFY WZLL 30494). All the animal experiments conform to the ARRIVE guidelines.

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