General Guidelines siRNA targeted sequence is usually 21 nt in length. Avoid regions within 50-100 bp of the start codon and the termination codon Avoid intron regions Avoid stretches of 4 or more bases such as AAAA, CCCC Avoid regions with GC content 30% or 60%. Avoid repeats and low complex sequence Avoid single nucleotide polymorphism (SNP) sites Perform BLAST homology search to avoid off-target effects on other genes or sequences Always design negative controls by scrambling targeted siRNA sequence. The control RNA should have the same length and nucleotide composition as the siRNA but have at least 4-5 bases mismatched to the siRNA. Make sure the scrambling will not create new homology to other genes. Tom Tuschl s rules Select targeted region from a given cDNA sequence beginning 50-100 nt downstream of start condon First search for 23-nt sequence motif AA(N sub 19). If no suitable sequence is found, then, Search for 23-nt sequence motif NA(N sub 21) and convert the 3 end of the sense siRNA to TT Or search for NAR(N sub 17)YNN Target sequence should have a GC content of around 50%A = Adenine; T = Thymine; R = Adenine or Guanine (Purines); Y = Thymine or Cytosine (Pyrimidines); N = Any. Rational siRNA design By experimentally analyzing the silencing efficiency of 180 siRNAs targeting the mRNA of two genes and correlating it with various sequence features of individual siRNAs, Reynolds et al at Dharmacon, Inc identified eight characteristics associated with siRNA functionality. These characteristics are used by rational siRNA design algorithm to evaluate potential targeted sequences and assign scores to them. Sequences with higher scores will have higher chance of success in RNAi. The table below lists the 8 criteria and the methods of score assignment.Criteria Description Score YesNo 1Moderate to low (30%-52%) GC Content1 point 2At least 3 A/Us at positions 15-19 (sense)1 point /per A or U 3Lack of internal repeats (Tm* 20 C)1 point 4A at position 19 (sense)1 point 5A at position 3 (sense)1 point 6U at position 10 (sense)1 point 7No G/C at position 19 (sense)-1 point 8No G at position 13 (sense)-1 pointA sum score of 6 defines the cutoff for selecting siRNAs. All siRNAs scoring higher than 6 are acceptable candidates.*Tm = 79.8 + 18.5*log sub 10([Na+ ] ) + (58.4 * GC%/100) + (11.8 * (GC%/100)2 ) - (820/Length)For example, the Tm can be calculated as follows for the siRNA UUCUCCAGCUUCUAAAAUATm = 79.8 + 18.5*log sub 10(0.05) + (58.4 * 31.6/100) + (11.8 * (31.6/100)2 ) - (820/19)Tm = 32.19 There are two siRNA design tools which implement this siRNA design algorithm: one is offered by Dharmacon, Inc; the other is a downloadable Excel template, written by Maurice Ho at http://boz094.ust.hk/RNAi/siRNA.References Elbashir SM et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411:494-498. Elbahir SM et al. (2001). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877-6888. Elbashir SM et al. (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods. 26:199-213. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 Mar;22(3):326-30. http://www.basic.northwestern.edu/biotools/oligocalc.html Maurice Ho, Rational siRNA Design 超方便的实验干货查询工具 微信扫码进入「丁香实验」小程序 查看全部 来源：互联网 超方便的实验干货查询工具微信扫码进入「丁香实验」 知道了 你可能喜欢 师弟问了我 25 个引物相关的问题