Optimization of cationic polymer-mediated transfection for RNA interference

Transfection efficiency was estimated to optimize the conditions for RNA interference (RNAi), including transfection time, validity, and nucleic acid concentration and type, using the EZ Trans Cell Reagent, a cationic polymer. An shRNA against GFP was designed and transfected into cells using the EZ transfection reagent. The shRNA significantly decreased the expression of GFP. In addition, pre-diluted transfection reagent at room temperature and small nucleic acids increased the transfection efficiency, which peaked at 24 h. Compared with circular nucleic acids, linear nucleic acids showed higher transfection efficiency and a higher genome integration rate. We optimized cationic polymer-mediated RNAi conditions, and our data will be useful for future RNAi studies.

Keywords:
shRNA; transfection reagent; cationic polymer; RNAi.

Authorship

Xiaojie Fan

Huaihe Hospital of Henan University, Kaifeng, Henan, China.Huaihe Hospital of Henan UniversityChinaKaifeng, Henan, ChinaHuaihe Hospital of Henan University, Kaifeng, Henan, China.

* These authors have contributed equally to this work.

Jingnan Yang

Huaihe Hospital of Henan University, Kaifeng, Henan, China.Huaihe Hospital of Henan UniversityChinaKaifeng, Henan, ChinaHuaihe Hospital of Henan University, Kaifeng, Henan, China.

* These authors have contributed equally to this work.

Guangyao Wu

Huaihe Hospital of Henan University, Kaifeng, Henan, China.Huaihe Hospital of Henan UniversityChinaKaifeng, Henan, ChinaHuaihe Hospital of Henan University, Kaifeng, Henan, China.

Meiyi Wang

Henan University, School of Medicine, Kaifeng, Henan, China.Henan UniversityChinaKaifeng, Henan, ChinaHenan University, School of Medicine, Kaifeng, Henan, China.

Xiaoxia Cheng

Henan University, School of Medicine, Kaifeng, Henan, China.Henan UniversityChinaKaifeng, Henan, ChinaHenan University, School of Medicine, Kaifeng, Henan, China.

Chang Liu

Henan University, School of Medicine, Kaifeng, Henan, China.Henan UniversityChinaKaifeng, Henan, ChinaHenan University, School of Medicine, Kaifeng, Henan, China.

Qian Liu

Henan University, School of Medicine, Kaifeng, Henan, China.Henan UniversityChinaKaifeng, Henan, ChinaHenan University, School of Medicine, Kaifeng, Henan, China.

Yanan Wen

Huaihe Hospital of Henan University, Kaifeng, Henan, China.Huaihe Hospital of Henan UniversityChinaKaifeng, Henan, ChinaHuaihe Hospital of Henan University, Kaifeng, Henan, China.

Shuangshuang Meng

Huaihe Hospital of Henan University, Kaifeng, Henan, China.Huaihe Hospital of Henan UniversityChinaKaifeng, Henan, ChinaHuaihe Hospital of Henan University, Kaifeng, Henan, China.

Zhenxing Wang

Huaihe Hospital of Henan University, Kaifeng, Henan, China.Huaihe Hospital of Henan UniversityChinaKaifeng, Henan, ChinaHuaihe Hospital of Henan University, Kaifeng, Henan, China.

Xuhong Lin

Huaihe Hospital of Henan University, Kaifeng, Henan, China.Huaihe Hospital of Henan UniversityChinaKaifeng, Henan, ChinaHuaihe Hospital of Henan University, Kaifeng, Henan, China.

Lei An

Huaihe Hospital of Henan University, Kaifeng, Henan, China.Huaihe Hospital of Henan UniversityChinaKaifeng, Henan, ChinaHuaihe Hospital of Henan University, Kaifeng, Henan, China.

Henan University, School of Medicine, Kaifeng, Henan, China.Henan UniversityChinaKaifeng, Henan, ChinaHenan University, School of Medicine, Kaifeng, Henan, China.

http://orcid.org/0000-0003-1072-542X

Send correspondence to
Lei An. Huaihe Hospital of Henan University, Ximen Avenue, No.115, Kaifeng, Henan, China. E-mail: hndxal@163.com.

Associate Editor:

Carlos F. M. Menck

*
These authors have contributed equally to this work.

Conflict of interest

None of the authors have a conflict of interest to declare.

Author Contributions

LA and XF conceived the study; JY, YW, SM, ZW, CL, QL and GW conducted the experiments; XC and JY analyzed the data; MW and LA wrote the manuscript. All authors read and approved the final version.

SCIMAGO INSTITUTIONS RANKINGS

Huaihe Hospital of Henan University, Kaifeng, Henan, China.Huaihe Hospital of Henan UniversityChinaKaifeng, Henan, ChinaHuaihe Hospital of Henan University, Kaifeng, Henan, China.

Henan University, School of Medicine, Kaifeng, Henan, China.Henan UniversityChinaKaifeng, Henan, ChinaHenan University, School of Medicine, Kaifeng, Henan, China.

Figures

Figures (7)

Thumbnail

Figure 1 -
In the GFP, single small nucleic acids, mixed GFP + NC, mixed GFP + small nucleic acids, and shRNA groups, transfection of the PMD-18T recombinant plasmid encoding GFP-shRNA significantly reduced GFP expression at 14, 24, and 48 h, with a nadir at 24 h.

Thumbnail

Figure 2 -
Fluorescence intensities of GFP-positive cells. (A) Fluorescence intensities of GFP-positive cells in the GFP, shRNA, GFP+NC, and GFP+shRNA groups at 14, 24, and 48 h (***p < 0.001). (B) Fluorescence intensities under the non-diluted, pre-diluted at 4 °C, and pre-diluted at room temperature conditions at 14, 24, and 48 h; ***p < 0.001; ns, not significant). (C) Student’s t-test for the comparison of 0.1 and 0.2 µg GFP at 14, 24, and 48 h; ***p < 0.001. (D) Fluorescence intensities in the control, mixed at 4 °C or room temperature without dilution, and mixed at 4 °C or room temperature with dilution groups (***p < 0.001). (E) Student’s t-test for comparison of the linear and circular small nucleic acids at 14 and 24 h. ns, not significant; **p < 0.01 and ***p < 0.001.

Thumbnail

Figure 3 -
Among the non-diluted, pre-diluted at 4 °C, and pre-diluted at room temperature conditions, the transfection reagent diluted at room temperature showed the greatest transfection efficiency at 14, 24, and 48 h.

Thumbnail

Figure 4 -
There were few GFP-positive cells after transfection of the GFP plasmid at 10 (0.1 g) - and 5 (0.2 g) - fold dilutions at 14, 24, and 48 h.

Thumbnail

Figure 5 -
GFP was used as the simulant. The premixed solution was prepared, and half of the solution volume was diluted with PBS. Next, one quarter volumes of the diluted and non-diluted solutions were stored at 4 °C and another quarter volume at room temperature. After storage, the cells were transfected and analyzed after 14, 24, and 48 h.

Thumbnail

Figure 6 -
Using GFP as the target, linear and circular small nucleic acids were used for interference, and different gradients were used to compare the effects under the same mass and the same mole number. Transfection efficiency was analyzed at 14 and 24 h.

Thumbnail

Figure 7 -
Genomic DNA was extracted, and genome insertion events were detected at 14 and 24 h.

Figure 1 - In the GFP, single small nucleic acids, mixed GFP + NC, mixed GFP + small nucleic acids, and shRNA groups, transfection of the PMD-18T recombinant plasmid encoding GFP-shRNA significantly reduced GFP expression at 14, 24, and 48 h, with a nadir at 24 h.

Figure 2 - Fluorescence intensities of GFP-positive cells. (A) Fluorescence intensities of GFP-positive cells in the GFP, shRNA, GFP+NC, and GFP+shRNA groups at 14, 24, and 48 h (p < 0.001). (B) Fluorescence intensities under the non-diluted, pre-diluted at 4 °C, and pre-diluted at room temperature conditions at 14, 24, and 48 h; p < 0.001; ns, not significant). (C) Student’s t-test for the comparison of 0.1 and 0.2 µg GFP at 14, 24, and 48 h; p < 0.001. (D) Fluorescence intensities in the control, mixed at 4 °C or room temperature without dilution, and mixed at 4 °C or room temperature with dilution groups (p < 0.001). (E) Student’s t-test for comparison of the linear and circular small nucleic acids at 14 and 24 h. ns, not significant; p < 0.01 and *p < 0.001.

Figure 3 - Among the non-diluted, pre-diluted at 4 °C, and pre-diluted at room temperature conditions, the transfection reagent diluted at room temperature showed the greatest transfection efficiency at 14, 24, and 48 h.

Figure 4 - There were few GFP-positive cells after transfection of the GFP plasmid at 10 (0.1 g) - and 5 (0.2 g) - fold dilutions at 14, 24, and 48 h.

Figure 5 - GFP was used as the simulant. The premixed solution was prepared, and half of the solution volume was diluted with PBS. Next, one quarter volumes of the diluted and non-diluted solutions were stored at 4 °C and another quarter volume at room temperature. After storage, the cells were transfected and analyzed after 14, 24, and 48 h.

Figure 6 - Using GFP as the target, linear and circular small nucleic acids were used for interference, and different gradients were used to compare the effects under the same mass and the same mole number. Transfection efficiency was analyzed at 14 and 24 h.

Figure 7 - Genomic DNA was extracted, and genome insertion events were detected at 14 and 24 h.

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Optimization of cationic polymer-mediated transfection for RNA interference

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[1] Send correspondence to
Lei An. Huaihe Hospital of Henan University, Ximen Avenue, No.115, Kaifeng, Henan, China. E-mail: hndxal@163.com.