I want to insert shRNA sequence to a vector with U6 promoter. What's the order of shRNA sequence? 5'-U6-sense-loop-antisense-terminator-3' or 5'-U6-antisense-loop-sense-terminator-3'? Is the sequence of the shRNA sense strand the same as target sequence (I found the target sequence according to the mRNA sequence in NCBI gene bank)?
Usually it is 5'-promoter-sense-loop-antisense-terminator-3'. Sequence of shRNA sense strand essentially follows the target sequence but may contain several mismatches because it should not bind too strong to the antisense strand (so, effectively these are the mismatches between shRNA sense and antisense strands, which makes the sense strand to have mismatches with the target). In its own turn, the antisense strand may also be designed to have some imperfect complementarity to the target if you want to hit several targets simultaneously or, vice versa, to decrease counterproductive pairing of the antisense strand with sequences other than the target.
Thanks, Victor. Your answer is very helpful. I thought the sequence should be 5'-promoter-sense-loop-antisense-terminator-3', and tried to design the insert according to it. However, after I read a protocol named "shRNA shagging", I got confused. The sequence of the insert which is the PCR product using the primers they design has an opposite sense antisense sequence.
The orientation may be not of particular importance. Since inside the cell the shRNA will be processed by Dicer, the loop will be chopped off and after that it does not matter how the strands were oriented in the original expression construct. However, people mostly use the 5'-promoter-sense-loop-antisense-terminator-3' layout, so it may be safer to follow the majority rule.
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