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RNA interference

Operation method

shRNA design

Materials and Instruments

【Materials】Target Cells, Vectors

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1. The shRNA cloned into the shRNA expression vector consists of two short inverted repeats separated by a loop, forming a hairpin structure controlled by the pol III promoter. This is followed by 5-6 T's attached as the transcription terminator of RNA polymerase III.2. Two complementary oligonucleotides must have restriction sites at both ends.3. Stratagene found that 29 oligonucleotides can repress the target gene more effectively than the 23 oligonucleotides originally recommended.4. A C is attached immediately below the cleavage site downstream of the promoter, spacing the insertion fragment from the promoter to ensure that transcription occurs.5. The first base of the ShRNA destination sequence must be a G to ensure transcription by RNA polymerase. If the selected destination sequence does not begin with a G, a G must be added immediately upstream of the justice strand.6. The stem-loop in the shRNA insert fragment should be near the center of the oligonucleotide. Stem loops of different sizes and nucleotide sequences have been used successfully. Stem loops containing a unique restriction site are favorable for the detection of clones with shRNA insert fragments. After comparing numerous stem loops of different lengths and sequences, the 5'TCAAGAG3' sequence was the most effective.7. 5-6 T's must be placed at the end of the shRNA insert fragment to ensure that RNA polymerase III terminates transcription.8. 3 or more consecutive T's must not be present on both the positive and antisense strand sequences. this may result in premature termination of shRNA transcription.

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