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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 did not affect the decay of renilla luciferase mRNA transcribed from pRL-SV40.(A) Curcumin significantly increased renilla luciferase mRNA levels compared with DMSO (*P < 0.05). (B) Two hundred nM of FP completely abrogated the curcumin-mediated increase in renilla luciferase mRNA levels (NS, not statistically significant). The renilla luciferase mRNA level was normalized to that of ACTB and subsequently normalized to 1 relative to the control treatment with DMSO. The data in all of the bar graphs were plotted as the mean ± SEM.
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f6: Curcumin did not affect the decay of renilla luciferase mRNA transcribed from pRL-SV40.(A) Curcumin significantly increased renilla luciferase mRNA levels compared with DMSO (*P < 0.05). (B) Two hundred nM of FP completely abrogated the curcumin-mediated increase in renilla luciferase mRNA levels (NS, not statistically significant). The renilla luciferase mRNA level was normalized to that of ACTB and subsequently normalized to 1 relative to the control treatment with DMSO. The data in all of the bar graphs were plotted as the mean ± SEM.

Mentions: To examine the effect of curcumin on renilla luciferase mRNA levels when the renilla luciferase gene lies downstream of the SV40 early enhancer/promoter, qPCR was carried out on pRL-SV40-transfected A549 cells treated with curcumin or DMSO. In agreement with the data from the luciferase reporter assay, qPCR revealed that, compared with DMSO, curcumin significantly increased renilla luciferase mRNA levels in A549 cells transfected with pRL-SV40 (Fig. 6A). To further determine the contribution of RNA decay to renilla luciferase transcript levels in A549 cells treated with curcumin, flavopiridol (FP) was used to block gene transcription, and qPCR was employed to determine renilla luciferase mRNA levels. The results demonstrated that 200 nM of FP completely abrogated the enhancement of renilla luciferase mRNA levels by curcumin (Fig. 6B), despite the fact that it significantly inhibited cell proliferation when combined with curcumin (Supplementary Fig. S1A,B). In addition, FP caused a time-dependent decrease in renilla luciferase mRNA levels (Supplementary Fig. S1C), which indicates that FP blocks the transcription of renilla luciferase. This is in line with previous reports showing that FP is a potent inhibitor of gene transcription910.


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 did not affect the decay of renilla luciferase mRNA transcribed from pRL-SV40.(A) Curcumin significantly increased renilla luciferase mRNA levels compared with DMSO (*P < 0.05). (B) Two hundred nM of FP completely abrogated the curcumin-mediated increase in renilla luciferase mRNA levels (NS, not statistically significant). The renilla luciferase mRNA level was normalized to that of ACTB and subsequently normalized to 1 relative to the control treatment with DMSO. The data in all of the bar graphs were plotted as the mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664949&req=5

f6: Curcumin did not affect the decay of renilla luciferase mRNA transcribed from pRL-SV40.(A) Curcumin significantly increased renilla luciferase mRNA levels compared with DMSO (*P < 0.05). (B) Two hundred nM of FP completely abrogated the curcumin-mediated increase in renilla luciferase mRNA levels (NS, not statistically significant). The renilla luciferase mRNA level was normalized to that of ACTB and subsequently normalized to 1 relative to the control treatment with DMSO. The data in all of the bar graphs were plotted as the mean ± SEM.
Mentions: To examine the effect of curcumin on renilla luciferase mRNA levels when the renilla luciferase gene lies downstream of the SV40 early enhancer/promoter, qPCR was carried out on pRL-SV40-transfected A549 cells treated with curcumin or DMSO. In agreement with the data from the luciferase reporter assay, qPCR revealed that, compared with DMSO, curcumin significantly increased renilla luciferase mRNA levels in A549 cells transfected with pRL-SV40 (Fig. 6A). To further determine the contribution of RNA decay to renilla luciferase transcript levels in A549 cells treated with curcumin, flavopiridol (FP) was used to block gene transcription, and qPCR was employed to determine renilla luciferase mRNA levels. The results demonstrated that 200 nM of FP completely abrogated the enhancement of renilla luciferase mRNA levels by curcumin (Fig. 6B), despite the fact that it significantly inhibited cell proliferation when combined with curcumin (Supplementary Fig. S1A,B). In addition, FP caused a time-dependent decrease in renilla luciferase mRNA levels (Supplementary Fig. S1C), which indicates that FP blocks the transcription of renilla luciferase. This is in line with previous reports showing that FP is a potent inhibitor of gene transcription910.

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