We are planning to transfect HEK293T cells with plasmid that carries shRNA sequence against the gene of interest, grow a large batch and let them produce shRNAs, and then isolate shRNAs. But the problem is we don't want miRNAs/shRNAs together cause that wouldn't appropriate for our hypothesis since we only want to see shRNA inhibition agaist the selected gene. So is there a kit, protocol or an appliance for extracting shRNAs from other RNAs including mirnas from cells?
Hi
You can design a strategy for isolation of desired shRNA, and their separation from other small RNAs from the total pool. Design an oligonucleotide partially complementary to the shRNA sequence. During synthesis get it modified to contain a 'biotin' group at 5'-end. Mix this with the total small RNA pool from the tissue sample and then use streptavidin coated tubes to fish out the duplex RNA (shRNA:biotin probe). This can be then used to any analysis including estimation of its levels. It should work, as similar kits for other purposes are available
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