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Evidence for transcriptional interference in a dual-luciferase reporter system.

Wu GQ, Wang X, Zhou HY, Chai KQ, Xue Q, Zheng AH, Zhu XM, Xiao JY, Ying XH, Wang FW, Rui T, Xu LY, Zhang YK, Liao YJ, Xie D, Lu LQ, Huang DS - Sci Rep (2015)

Bottom Line: Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs.We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups.These results explain the discrepancies between the two luciferase reporter systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.

ABSTRACT
The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3'untranslated region (3'UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3'UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems.

No MeSH data available.


Related in: MedlinePlus

Curcumin promoted the transcription of the luciferase gene located downstream of the SV40 early enhancer/promoter.(A–C) Curcumin treatment increased renilla luciferase activity relative to DMSO treatment when the absolute copy number of the dual-luciferase reporter vector p1 successfully transfected into A549 cells was used as an internal control (A, *P < 0.05). Luciferase activity (hRluc:absolute copy number of vector) was normalized to 1 relative to the control treatment with DMSO. The absolute renilla luciferase activity derived from A549 cells treated with curcumin was higher than that derived from A549 cells treated with DMSO (B, *P < 0.05). The absolute copy number of the dual-luciferase reporter vector p1 successfully transfected into A549 cells treated with curcumin was lower than that of the A549 cells treated with DMSO (C, *P < 0.05). (D–F) When pRL-SV40 was used as the internal control, the absolute renilla luciferase activity derived from A549 cells treated with curcumin was higher than that derived from A549 cells treated with DMSO (D, *P < 0.05). When pRL-TK or pRL-CMV was used as the internal control, the absolute renilla luciferase activity derived from A549 cells treated with curcumin was lower than that derived from A549 cells treated with DMSO (E and F, *P < 0.05). The data in all of the bar graphs were plotted as the mean ± SEM.
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f4: Curcumin promoted the transcription of the luciferase gene located downstream of the SV40 early enhancer/promoter.(A–C) Curcumin treatment increased renilla luciferase activity relative to DMSO treatment when the absolute copy number of the dual-luciferase reporter vector p1 successfully transfected into A549 cells was used as an internal control (A, *P < 0.05). Luciferase activity (hRluc:absolute copy number of vector) was normalized to 1 relative to the control treatment with DMSO. The absolute renilla luciferase activity derived from A549 cells treated with curcumin was higher than that derived from A549 cells treated with DMSO (B, *P < 0.05). The absolute copy number of the dual-luciferase reporter vector p1 successfully transfected into A549 cells treated with curcumin was lower than that of the A549 cells treated with DMSO (C, *P < 0.05). (D–F) When pRL-SV40 was used as the internal control, the absolute renilla luciferase activity derived from A549 cells treated with curcumin was higher than that derived from A549 cells treated with DMSO (D, *P < 0.05). When pRL-TK or pRL-CMV was used as the internal control, the absolute renilla luciferase activity derived from A549 cells treated with curcumin was lower than that derived from A549 cells treated with DMSO (E and F, *P < 0.05). The data in all of the bar graphs were plotted as the mean ± SEM.

Mentions: The two different luciferase reporter systems generated contradictory data regarding the effects of curcumin on luciferase reporter expression. To reconcile this discrepancy, we focused on the dual-luciferase reporter vector pmiR-RB-REPORTTM. Based on the analysis of the features of this vector, several possibilities were proposed to explain the inconsistent results. First, we speculated that the activity of the internal control firefly luciferase in p1 was suppressed by curcumin, which makes it unable to serve as an internal control. To test this hypothesis, quantitative real-time PCR (qPCR) was employed to determine the absolute copy number of the dual-luciferase reporter construct p1 that was successfully transfected into the A549 cells. The specific activity of EZH2 3′UTR-renilla luciferase in cells treated with curcumin was compared to that in cells treated with DMSO by normalizing to the absolute copy number of the internal control plasmid. In agreement with the results generated by using firefly luciferase activity as the internal control, the EZH2 3′UTR-renilla luciferase activity derived from the dual-luciferase reporter construct p1 was higher in cells treated with curcumin compared to those treated with DMSO (Fig. 4A). This indicates that curcumin likely does not affect the expression of the firefly luciferase gene driven by the herpes simplex virus thymidine kinase (HSV-TK) promoter in p1.


Evidence for transcriptional interference in a dual-luciferase reporter system.

Wu GQ, Wang X, Zhou HY, Chai KQ, Xue Q, Zheng AH, Zhu XM, Xiao JY, Ying XH, Wang FW, Rui T, Xu LY, Zhang YK, Liao YJ, Xie D, Lu LQ, Huang DS - Sci Rep (2015)

Curcumin promoted the transcription of the luciferase gene located downstream of the SV40 early enhancer/promoter.(A–C) Curcumin treatment increased renilla luciferase activity relative to DMSO treatment when the absolute copy number of the dual-luciferase reporter vector p1 successfully transfected into A549 cells was used as an internal control (A, *P < 0.05). Luciferase activity (hRluc:absolute copy number of vector) was normalized to 1 relative to the control treatment with DMSO. The absolute renilla luciferase activity derived from A549 cells treated with curcumin was higher than that derived from A549 cells treated with DMSO (B, *P < 0.05). The absolute copy number of the dual-luciferase reporter vector p1 successfully transfected into A549 cells treated with curcumin was lower than that of the A549 cells treated with DMSO (C, *P < 0.05). (D–F) When pRL-SV40 was used as the internal control, the absolute renilla luciferase activity derived from A549 cells treated with curcumin was higher than that derived from A549 cells treated with DMSO (D, *P < 0.05). When pRL-TK or pRL-CMV was used as the internal control, the absolute renilla luciferase activity derived from A549 cells treated with curcumin was lower than that derived from A549 cells treated with DMSO (E and F, *P < 0.05). The data in all of the bar graphs were plotted as the mean ± SEM.
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Related In: Results  -  Collection

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f4: Curcumin promoted the transcription of the luciferase gene located downstream of the SV40 early enhancer/promoter.(A–C) Curcumin treatment increased renilla luciferase activity relative to DMSO treatment when the absolute copy number of the dual-luciferase reporter vector p1 successfully transfected into A549 cells was used as an internal control (A, *P < 0.05). Luciferase activity (hRluc:absolute copy number of vector) was normalized to 1 relative to the control treatment with DMSO. The absolute renilla luciferase activity derived from A549 cells treated with curcumin was higher than that derived from A549 cells treated with DMSO (B, *P < 0.05). The absolute copy number of the dual-luciferase reporter vector p1 successfully transfected into A549 cells treated with curcumin was lower than that of the A549 cells treated with DMSO (C, *P < 0.05). (D–F) When pRL-SV40 was used as the internal control, the absolute renilla luciferase activity derived from A549 cells treated with curcumin was higher than that derived from A549 cells treated with DMSO (D, *P < 0.05). When pRL-TK or pRL-CMV was used as the internal control, the absolute renilla luciferase activity derived from A549 cells treated with curcumin was lower than that derived from A549 cells treated with DMSO (E and F, *P < 0.05). The data in all of the bar graphs were plotted as the mean ± SEM.
Mentions: The two different luciferase reporter systems generated contradictory data regarding the effects of curcumin on luciferase reporter expression. To reconcile this discrepancy, we focused on the dual-luciferase reporter vector pmiR-RB-REPORTTM. Based on the analysis of the features of this vector, several possibilities were proposed to explain the inconsistent results. First, we speculated that the activity of the internal control firefly luciferase in p1 was suppressed by curcumin, which makes it unable to serve as an internal control. To test this hypothesis, quantitative real-time PCR (qPCR) was employed to determine the absolute copy number of the dual-luciferase reporter construct p1 that was successfully transfected into the A549 cells. The specific activity of EZH2 3′UTR-renilla luciferase in cells treated with curcumin was compared to that in cells treated with DMSO by normalizing to the absolute copy number of the internal control plasmid. In agreement with the results generated by using firefly luciferase activity as the internal control, the EZH2 3′UTR-renilla luciferase activity derived from the dual-luciferase reporter construct p1 was higher in cells treated with curcumin compared to those treated with DMSO (Fig. 4A). This indicates that curcumin likely does not affect the expression of the firefly luciferase gene driven by the herpes simplex virus thymidine kinase (HSV-TK) promoter in p1.

Bottom Line: Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs.We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups.These results explain the discrepancies between the two luciferase reporter systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.

ABSTRACT
The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3'untranslated region (3'UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3'UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems.

No MeSH data available.


Related in: MedlinePlus