Hi everybody,
we are working with let-7 family and we would like to perform knock-down of the let-7 using Lin28 expression vector. In another experiment we would like to over-express let-7g from a vector.
We have purchased MSCV Lin-28 and let-7g expressing vectors from Addgene. We validated vectors using sequencing and they look fine.
Currently, we are performing experiments using 3T3 cells and we are using electroporation to deliver the vector. We have optimised the electroporation conditions using GFP expressing vector. Additionally, we are monitoring Lin-28 expression by qPCR after transfection.
Problem starts when we want to quantify changes in let-7g expression. Although, Lin-28 is highly expressed (24h after transfection) we don't see change in let-7g expression. Same thing occurs when we perform let-7g vector transfection. We monitor after the transfection the expression of the known let-7 target (Myc) and we cannot detect any change in target expression either.
For miRNA isolation we use Trizol and for detection we use miScript kit (Qiagen) and to make sure we do not have a problem with detection we performed a spike-in experiment. We added let-7g RNA oligo to both Trizol and to isolated RNA and perform RT and qPCR. We can perfectly detect different spike-in concentrations.
Currently, we are trying to isolate RNA at different time points after transfection. We tried 24 h and 72 h. We still cannot detect any change.
We are quite confused about the issue and would highly welcome any suggestion.
Thank you in advance.
problems seem the binding between lin28 and let7; I would suggest controlling again the DNA sequence in order to exclude you have some mutant and to repeat the experiment using another cell line!
We have solved the lin28 issue so I will post the solution hoping it help somebody else. Stable transfection of lin28 vector in 3T3 cell line did the trick. It seems it is necessary that lin28 is expressed for prolonged time at high levels in order to downregulate let-7 family. After 15 days expression of all members fell down to only 10-15% of original expression.
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