I am planning to use pSuper vector system for over-expressing and knocking down a microRNA in breast cancer cells. Although it was initially designed for the purpose of siRNA expression, I am not quite sure about its suitability as far as miRNA expression is concerned. Also, I also would like to know that whether cloning a sequence antisense to a particular miRNA in the same system can efficiently result in knockdown of that miRNA.
Hi Deepti,
I work with miRNA and to over-express, a particular miRNA first I amplified ~350 bp region containing pre-miRNA with my desired restriction site on both ends. I cloned this into pGEMT-Easy vector and then I cloned that piece into the PCDNA 3.1 + vector (zeocin). I transfected HCC-1954 cells with this cloned vector (0.5 ugs) and collected around 25 individual clones. It is extremely important to collect more clones since expression of your miRNA will vary from clone to clone. Based on Real time data, I have approximately 800-4000 fold up regulation of my miRNA of interest. For knock down the most suitable approach will be to use sponge. see this paper http://www.jbc.org/content/early/2013/08/06/jbc.M113.491803.full.pdf.
I hope this helps,
Good luck
Nirav
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