RNAi技术讨论
丁香园论坛
1319
1.在设计RNAi实验时，可以先在以下网站进行目标序列的筛选： http://www.ambion.com/techlib/misc/siRNA_finder.html http://katahdin.cshl.org:9331/RNAi/ http://www.ic.sunysb.edu/Stu/shilin/rnai.html
2.RNAi目标序列的选取原则： （1）siRNAs with lower G/C content (35-55%) are more active than those with G/C content higher than 55%； （2）Beginning with the AUG start codon of your transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl, et al. recommend against designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex. （3）Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences.
3.siRNA的合成： (1)IN VITRO: siRNAs generated in vitro by transcription with T7 DNA polymerase; (2)IN VIVO: Short hairpin RNAs that are transcribed in vivo from vectors containing the human U6 promoter.
4.目前已证实的siRNA可以在下面的网页找到： http://www.dharmacon.com/4DCGI/WEB_Menu/87978323/3310/7/4DWPG_11054394851
2.RNAi目标序列的选取原则： （1）siRNAs with lower G/C content (35-55%) are more active than those with G/C content higher than 55%； （2）Beginning with the AUG start codon of your transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl, et al. recommend against designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex. （3）Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences.
3.siRNA的合成： (1)IN VITRO: siRNAs generated in vitro by transcription with T7 DNA polymerase; (2)IN VIVO: Short hairpin RNAs that are transcribed in vivo from vectors containing the human U6 promoter.
4.目前已证实的siRNA可以在下面的网页找到： http://www.dharmacon.com/4DCGI/WEB_Menu/87978323/3310/7/4DWPG_11054394851
