Protocol for ShRNA generation____ utilizing PRS system by felix xu.doc

可在线设计shRNA的网站 【01】/bioc/siRNA/home.php 【02】/techlib/misc/siRNA_finder.html【03】/rnadesign/default.aspx?SID【04】/Order_Entry/jsp/ShowOptions.jsp?Country=10Proceed=Proceed【05】/rnaidesigner/【06】:9331/RNAi/html/rnai.html【07】/Stu/shilin/rnai.html【08】/business/products/order2.htm【09】/sirna.pl【10】/rnai/Basics: The basics of RNAi mechanism are shown in the cartoon below. To improve the RNAi efficiency, various systems are developed including using shRNA: For detailed information, see review: RNA interference and the use of small interfering RNA to study gene function in mammalian systems. Protocol: Strategy: Define target sequence: There are at least 2 ways to determine your target sequence: do it yourself or refer to published references; A: Do it yourself I recommend two databases for your design: 1. /bioc/siRNAext/home.php 2. /techlib/misc/siRNA_finder.html B: refer to published references 如果是参考前人的话，要看好文章的质量，引用率，效率等参数，设计时最好在靶基因的不同位置找靶序列。 此外，文献查阅时可以留心别人的scramble sequence information。 Order your oligos compatible with PRS according to following manner Forward oligo: 5 gatctcc—19bp sense—ttcaagaga—19bp antisense—tttttggaac3 Reverse oligo: 5 tcgagttccaaaaa—19bp sense—tctcttgaa—19bp antisense—gga3 And they will be annealed like this: For example: The sequence selected for Mdm2 shRNA design: GCCATTGCTTTTGAAGTTA GAPDH CACTCAAGATTGTCAGCAA TTGCTGACAATCTTGAGTGAG SCRAMBLE TTCTCCGAACGTGTCACGT ACGTGACACGTTCGGAGAA CBX2-1 GCGCCATCCAAATCCAAAT ATTTGGATTTGGATGGCGC CBX2-2 GCTCCATCGTACACTACAT ATGTAGTGTACGATGGAGC CBX2-3 CTCCCACAGACAACAACAA TTGTTGTTGTCTGTGGGAG Cbx5-3 gcggTgacTTaaTgTTcTT AagaacaTTaagTcaccgc scramble gatctcc TTCTCCGAACGTGTCACGT ttcaagaga ACGTGACACGTTCGGAGAA tttttggaac tcgagttccaaaaa TTCTCCGAACGTGTCACGT tctcttgaa ACGTGACACGTTCGGAGAA gga CBX2-1 gatctcc GCGCCATCCAAATCCAAAT ttcaagaga ATTTGGATTTGGATGGCGC tttttggaac tcgagttccaaaaa GCGCCATCCAAATCCAAAT tctcttgaa ATTTGGATTTGGATGGCGC gga CBX2-2 gatctcc GCTCCATCGTACACTACAT ttcaagaga ATGT