I have shown that reporter protein level is reduced but I want to confirm my reporter mRNA is actually degraded in response to miRNA binding.
I believe most mRNA transfected to cells is trapped in endosomes and so bulk RT-qPCR analysis from cells may not be that accurate, as the mRNA will be protected from miRISC.
I have designed qPCR primers flanking the miRNA binding site of the mRNA and so this should be sensitive to show subtle changes in mRNA level by qPCR.
Of course, the mRNA levels might not be affected if the MOA here is translational inhibition only.
Why not try a perfect site instead of a miRNA one - a cleavage assay? This will possibly not answer whether the whole RISC machinery is active, but will give a good indication of whether the mRNA is accessible.
Right, actually my miRNA site is the 5'UTR so I think the main mechanism is the miRNA-mRNA binding prevents ribosome scanning from the 5' cap but I have to show experimental data to show there is no major change at the RNA level. I have confirmed with Cy5 labeled RNA, that a lot seems to be trapped in endosomes. This seems to be the same using either Stemfect, Messenger MAX, or TransIT lipofection reagents
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