Hi there,
I am trying to design primers to detect shRNA in the transfected cells with my vector. The structure of shRNA within the vector is stem-loop-stem, with a terminator downstream of it and a human promotor gene upstream. I selected different lengths of sequences in the bold area (below) and designed a few sets of flanking primers to include at least one stem region of the shRNA, but none of the primer sets showed any signal in qPCR. I am new to this area and feel I am doing something wrong. Can you please advise?
5'- Human promotor_T_Stem-Loop-Stem_C_Terminator -3'
Many thanks, Dejun Ma for your answer!
I wasn't familiar with this technique. I will dig into it.
Hello,
This type of qPCR quantification is quite routine now a days. Try to keep a positive control, which can be another shRNA (from lab colleagues or literature) known to be working after transfection. Also, the secondary structure should be resolved properly prior to reverse transcription (if involved)
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