Part:BBa_K3730012:Experience
To further verify the specificity of the virus, we also tested the function of the designed miRNA sponges. HepG2 were transfected with 3u and sponges (sponge-pcDNA3.1-P2A-C,or sp), and the controlled group was transfected with null sponge plasmid (sponge-empty-pcDNA3.1-P2A-C, or spe), the ratio of these two plasmids was from 1:1 to 1:4. The statistical analysis results were listed below in Table. 1 and Table. 2.
| Df | Sum Sq | Mean Sq | F value | Pr(>F) | |
|---|---|---|---|---|---|
| sp and spe | 1 | 1187040 | 1187040 | 20.9136 | 0.0005226*** |
| concentration gradient | 1 | 93174 | 93174 | 1.6416 | 0.2224950 |
| Residuals | 13 | 737869 | 56759 | ||
| Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1 |
Table. 1 LSD test results of the fluorescein HepG2 and Hek293T.
The significance was seen when the ratios of the two plasmids (sponge or sponge empty and 3u) were regarded as a covariate.
(3u refers to 3’UTR-pcDNA3.1-N-eGFP, sponge refers to sponge-pcDNA3.1-P2A-C, and sponge empty refers to sponge-empty-pcDNA3.1-P2A-C. )
| Difference | pvalue | signif. | LCL | UCL | |
|---|---|---|---|---|---|
| 3U+sp:1:1 - 3U+spe:1:1 | 384.491282 | 0.0399 | * | 20.55030 | 748.4323 |
| 3U+sp:1:3 - 3U+spe:1:3 | 705.022404 | 0.0011 | ** | 341.08142 | 1068.9634 |
Table. 2 LSD test results of the fluorescein HepG2 and Hek293T.
The significance was seen when the ratios of the two plasmids (3u and sponge or sponge empty ) were 1:3.
(3u refers to 3’UTR-pcDNA3.1-N-eGFP, sponge refers to sponge-pcDNA3.1-P2A-C, and sponge empty refers to sponge-empty-pcDNA3.1-P2A-C. )
Figure. 1 Fluorescence intensity after cotransfection of 3U and Sp/Spe in Hek293T and HepG2.
(3u refers to 3’UTR-pcDNA3.1-N-eGFP, sponge refers to sponge-pcDNA3.1-P2A-C, and sponge empty refers to sponge-empty-pcDNA3.1-P2A-C. )
According to Fig. 1 (a), in Hek293, when the ratio of the 3u and the pSp or pSp null is 1:3, the expression of the eGFP was significantly lowered. Fig. 1 (b) shows that in HepG2, the significance was shown also when the ratio was 1:3. LSD test also showed the same results.
Meanwhile, as shown in Table. 1, when the ratios of the two plasmids (sponge or sponge empty and 3u) were regarded as a covariate, the difference between the experimental group and the control group is significant, which indicates that sponges can increase the expressions of the eGFP.
However, as the sponges also increased the expression of that gene in Hek293T, which represents normal cells, we are bound to improve the specificity of the sponge parts.
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