Hi everyone. How soon after targeting b-Catenin with siRNA can we start to expect degradation of the RNA? Hours? Minutes? Trying to find answers for both in vitro and in vivo (xenograft) cancer experiments. Thanks.
This is an interesting question! You are planning knock-down experiments using interference of expresson by siRNA. It means that the siRNA will prevent de-novo synthesis of b-Catenin. I do not know how quickly the mRNA of b-Catenin breaks down. Certain transcripts are more stable than others. You also have to take into account the stability of existing b-Catenin protein. You certainly need to take time samples over a few hours starting from the time of induction of your siRNA (t=0) and measure both the protein levels (through WB) and mRNA (through RT-PCR) over time. Certain siRNA constructs will do better than others. Do not bet on one horse. Good luck.
Dear Oliver
In addition to Dr Jan Voskuil said, i wanted to add that in one in-vitro experiments, it has taken 3-5 days in decrease of 75-85% mRNA level of beta catenin but the decrease in beta catenin at mRNA level has been observed at day 1. Hence in absence of wnt, beta catenin gradually degrade. The beta catenin which has been observed during the initial phase of the experiment is the one which is already expressed and which is existing beta catenin protein inside the cell as Dr Jan told. I hope this will help.
Hi Oliver, we've used siPOOLs (high complexity siRNA pools from sitools biotech, www.sitoolsbiotech.com) against beta-catenin (CTNNB1) and saw 90% downregulation of RNA at 0.2nM by 24h in MCF7 cells. Hope this helps!
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