Part:BBa_K3734024

miR21T-LUC-4XmiR21T

CHREBP is a promoter that can be induced by glucose.And miR21T is miR21's target.When miR21 combined with miR21T, it will inhibit the expression of LUC. Luciferin(LUC) is a protein that can activate fluorescein.We use it as reporter gene.

miR21T-LUC-4XmiR21T

To prove miR21 can target miE21T and suppress the expression of the target gene, we have designed miR21T-LUC-4XmiR21T

1.Pattern diagram

Fig.1 The model diagram of miR21T-LUC-4XmiR21T

2.1 Method

We used LUC as report genes to reflect the level of expression through detecting luminescence value at 560nm wavelength and the error REN luminescence caused by the number of eliminated cells. In the mean time, we tested the level of mRNA expression through qPCR.

2.2 Result

Fig.2 Electrophoretic diagram of miR21T-LUC-4XmiR21T PCR product

At the time of the design experiment, the first 48-hour test reported that the gene LUC/REN ratio was reported, and miR21 inhibited 40% of the expression, but this was not ideal for our design.

Fig.3 miR21 inhibited Luc expression 48h after transfection

Considering that it may be the reason for the continuous accumulation of LUC expression, we did another 24-hour group, and the results proved that miR21 suppressed 90% of the expression, and the experimental results were very in line with our design requirements.

In the mean time, we have tested miR21 to speed up the degradation of mRNA through qPCR.

Fig.4 miR21 inhibited Luc expression 24h after transfection

Fig.5 miR21 promoted the mRNA degradation efficiency of LUC

3.Caution

Because we have repeated mE21T for 4 times at 3’UTR area, we should avoid the repeated pieces when designing the primer. Please pay attention to how many times does miR21T have repeated in PCR outcome. Meanwhile, there is similar parts between miR21T and padrome structure, which should be paid attention to during the design

Reference：

[1]Kai Zhang , Xue-Jiao Yang , Ting-Ting Zhang.In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.Anal ChimActa.2019Nov4;1079:146-152.