siRNA/RNAi/miRNA transfection human

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5 years ago

Keith L. Morrison

4

siRNA/RNAi/miRNA transfection human

I would like to regulate the expression of a gene and in order to do that, I have purchased specific siRNA. After optimizing my transfection protocol and using electroporation I have achieved a 60-70% reduction of the gene of interest. However, I cannot observe a significant reduction of mRNA expression but only a reduction of protein. What might be the problem? Could the problem be in my cell treatment method?

Experiment: siRNA / RNAi /miRNA transfection Human Cells - HESC Lipofectamine

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**Aaron Stege** · Answer 1 · 2019-11-14T21:04:10+00:00

You can exclude cell treatment being an issue by doing a mock transfection by following the same protocol without the use of siRNA. If there is no change in protein production then the cell treatment should be fine. Personally, I think the problem is the time of taking your measurements. The levels of mRNA and proteins fluctuate at different rates after the transfection of siRNA. mRNA knockdown occurs at 24-48hrs and then the mRNA levels may begin to return to normal. On the other hand, protein knockdown occurs at 48-96 hours since the siRNA is not targeting the proteins directly. If you think that you are analyzing your sample at the correct times then I would suggest that you use a different transfection reagent for siRNA that might be more efficient.

**Olivia Greene** · Answer 2 · 2021-06-16T07:14:41+00:00

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Answered 3 years ago

Olivia Greene

**Antonia N. Hanson** · Answer 3 · 2020-03-25T10:53:44+00:00

There's some evidence that miRNAs can block translation OR promote RNA decay. I think the former is pretty rare, but is a thing. Maybe your siRNA is functioning differently than you thought. Most people try multiple siRNAs for the same target and present data from 2+ different siRNA sequences, so my advice would be to try some more. It could also be that your primers are bad.

**Aaron Stege** · Answer 4 · 2019-11-14T21:04:10+00:00

You can exclude cell treatment being an issue by doing a mock transfection by following the same protocol without the use of siRNA. If there is no change in protein production then the cell treatment should be fine. Personally, I think the problem is the time of taking your measurements. The levels of mRNA and proteins fluctuate at different rates after the transfection of siRNA. mRNA knockdown occurs at 24-48hrs and then the mRNA levels may begin to return to normal. On the other hand, protein knockdown occurs at 48-96 hours since the siRNA is not targeting the proteins directly. If you think that you are analyzing your sample at the correct times then I would suggest that you use a different transfection reagent for siRNA that might be more efficient.