hRNA详细设计教程-以pLKO.1-Puro为载体设计.doc

shRNA设计此教程用于不知道siRNA靶点的设计，从文献中选择序列，可以从第5步开始设计1. 选择基因例如 Gene Name: TP53 Accession: NM_000546. 52. 寻找靶点http://rnaidesigner. thereofisher・ coiri/rneiiexpress/setOption. do?designOption二shrna&pid二-7168952604080645646输入基因序列号，或者粘贴序列AccBSsion number ni.L000546 日ORHudeotide sequence: Enter only A C. G T and U S«« th* online Help for additional information点击RNAi设计RNAi Design3. 靶点选择（选择星号比较多的2-3个作为目的序列）10shRNA Sequences (Upto 10 top sconng target sequences are reported sorted by tie Start postion and ranked as to to mdcateknockdo^ probaMity) Select the sequence to order and click MD«$ign shRMA Obgos*SelectNo.SUirt✓15372 683377840055973061048710508115391162GCTT CTT GCATT CTGGGACAG GCCATCTACAAGCAGTCACAG GCATCnATCCGAGTGGAAGG GCGTOTGGAOTATTTGGATGA GGAAGACTCCAGTGGTAATCT GCGCACAGAGGAAGAGAATC* GCACAGAGGAAGAGAATCTCC GCCAAAGAAGAAACCACTGGA GAAACCACTGGATGGAGAATAORFORTORFORFORF39523952C26239523962524786ORF 52 3910 1231 GCTGAATGAGGCCUGGAACTDesign shRNA OligosHote: As a result of the wc・nt update to BLOCK CT™ RflAi Cwgn” tht "Star Sconng System" has al&o been updated to reflect our rrwt highly refined RUAi dupkx designs Only the dupiexts with th< highest pcobJbilrty of success pcoMded Much means that out of ・ possible M stars no duplex has l«s$ than a thrtt-star ranking While each individual RNAi duplex i$ designed to achieve tht highest Qualty results wt recommend sekctmg th# top three star- raaked for your target of interest to guarantee your knockdown success4. shRNA网页设计点击设计shRNA OLigosDesign shRNA OligosDesigning shRHA OtogosS#hct one “Go default loop sequences o< specif/ a custom loop sequtnc« for each target sequence to Q^nerate shRriA obgos loc cloning mto irMrostn s BLOCK- Induobie RNAi Entry Vector (pENTR・/H[/TOL3 elected taraetso a" ecu ▼DesignReset Form网页上的loop可以不更改（后面需要更改为常用loop序列TTCAAGAGA）点击设计设计好的序列如下（上面是以thermo的载体（pENTR™/U6）设计）帖性末loop帖性末姑22 973 Top Strand s・・ cacc3*BonomStrand 5*- AAk；d$ Oligo 5•- CACCGGA^SPCCA5】GSTWCTCGNaGAmCaCNa6TCTTX -3*11 ii i ill i mi limn i ii 11 ill ill i ilium i ii ii i3•- CCTTCTGAGGTCACCX~A5A5CmCTAATGGT3A??TCXSAA3-3AAAA ・5,3 1048 Top Strand $•- »CCGCGCAOU»G<^ASAGAX：C：CGAJUGATTCTCTTCCTCI5TGC3C ・s・Bonom strand 51- AAXAXGCACX^SAAGXGAXTCmCGAGA~CTCn(XTCTGTGC3C -3*ds OI19O 51- CXCCGCGmMAZuSAk：二0AAX0eTTCCTCTGTGC3C -3fllllllllllllllllllllllllllllllllllllllllllllll3•- CGCGTGTCTCCTTCTCTTASAGCmCTAA3AGAX3GASACACXGAAAX -5*5. 后续的根据自己的载体手工设计shRNA（以PLKO. 1-puro设计,酶切位点Agel EcoRI,酶切位点后1300bp有一个单一的酶切位点Kpnl)Age!ttgtggaaaggacgaaacaccggtgaattctcgacctcgagacaaaacacctttcctgctttgtggccacttaagagctggagctctgtt> ▲ ▲注意：shRNA比较短，而且结构复杂，测序困难，选择载体上的单一酶切位点，设计•到shRNA里面，用于后续的单酶切验证设计模板按照下图（下图去除Agel和EcoRI,也可以适当位置添两个碱基保留看两个酶切位点）5' -CCGG siRNA 正向序列 TTCAAGAGA 反向互补序列 TTTTTT GGTACC-3'正向互补序列AAGTTCTCT 反向序列AAAAAA CCATGG TTAA-5?椅凶粘性末端常用loop转录终止Kpnl用于验证EcoRI粘性末端5' CCGG siRNA正向序列 头 正向互补序列TCAAGAGGTTCT反向互补序列TTTTTT GGTACC・3' * 反向宇列 AAAAAA CCATGG TI■•亍将网页上设计的shRNA序列替换到上述模板ds Oligo正向序列5f- CACCGCTTCTTGCATTCTGGGACAGCGAACTGTCCCAGAATGCAAGAAGC -3fllllllllllllllllllllllllllllllllllllllllllllll正向互补SE^TGACAGGGTCTTACGTTCTTCGAAAA -5f反向序列替换之后如下5' -CCGG GCTTCTTGCATTCTGGGACAGTTCAAGAGACTGTCCCAGAATGCAAGAAGCTTTTTT GGTACC-3，3' - CGAAGAACGTAAGACCCTGTCAAGTTCTCTGACAGGGTCTTACGTTCTTCGAAAAAA CCATGGTTM-5'注意：正义链为5, 一3,,反义链为:T -5,,合成时需要进行反向处理。

6. 验证设计的shRNA是否合适二级结构（用DNAMAN分析二级结构）DNAMAN • DNAMAN〔.Structure]File EditSequence | Search Restriction Primer Protein Database Info View Window HelpCurrent Channelload SequenceDisplay SequenceRot DNA PropertiesDraw Sequence Mapf DNAMAN1 用DNAMAN 1 9DNAMAN—Structure[3 DNAMAN1Secondary StructureCurrent SequenceSequence AssemblyAll Available Sequencesy o2 the itructur•-・42・70 kcal/aolStructure EnergyDot Matrix ComparisonAlignmentRandom Sequence分析正反向引物是否匹配ffl Snapgene分析，正向序列输入进去，反向的作为引物用來验证（注意5' 3'方向）関切位点Kpnl一个正确的shRNA应该入下图Pnmer 1LS △"乂爲 CTTCTTGCATTCTGGGACAG1^^^；CTGTCCCAGAATGCAAG^^^|39CC0G0CTTCTT0CATTCT000ACA0TTCAA6AGACTGTCCCAGAATGCAAGAAGCTTTTTT00TACC00CCC0AA0AAC0TAA6ACCCT0TCAA0TTCTCT0ACA0G0TCTTAC0TTCTTCGAAAAAACCAT0G39•75r粘性末端[COAAOAACOTAAOACCCTOTCAAOTTCTCTOACAOOOTCTTACOTTCTTCOAAAAAACCATOOyY^]Pnmer 17. 最终发送合成的序列为5' -CCGG GCTTCTTGCATTCTGGGACAGTTCAAGAGACTGTCCCAGAATGCAAGAAGCTTTTTT GGTACC-3'5' - AATTGGTACCAAAAAAGCTTCTTGCATTCTGGGACAGTCTCTTGAACTGTCCCAGAATGCAAGAAGC -3'8. 引物合成shRNA对引物纯度要求不高，不需要用page纯化，为了节约成本，用低一级的纯化方式HAP即可使用。

9. 引物退火连接稀释到50uM 取各取4. 5ul,加lul,退火buffer （可以用不含dNTP和酶的PCR buffer）, 95°C 3min, 口然降温到室温将载体酶切尽可能保证载体全部双酶切切开，可以选择NEB没有星活性的的酶取lul退火后的溶液，与载体（20-50ng就够了）连接并转化10 .质粒提取并验证如果有载体自连对照为阴性，可以取两个单克隆提质粒Kpnl单酶切，如果有能切出一个 1300bp左右的片段，可以认为构建是正确的取一个质粒进行测序，测序的时候告知测序公司，此质粒为发卡结构，和poly结构附录1・shRNA启动子多数的siRNA表达载体依赖RNA聚合酶III启动子（pol IID44的一种，操纵一段45— 50n。