The following recommendations for siRNA hairpin design and cloning strategy are made based on research by Ambion scientists.
Schematic of a Typical Hairpin siRNA Produced by an siRNA Expression Vector or an siRNA Expression Cassette and Its Relationship to the RNA Target Sequence.Īmbion's Recommended Procedure for siRNA Hairpin Design Prior to these experiments, each siRNA should be tested to ensure that it reduces target gene expression by comparable levels.įigure 1. Perhaps the best way to ensure confidence in RNAi data is to perform experiments, using a single siRNA at a time, with two or more different siRNAs targeting the same gene. Additional siRNA sequences targeting the same mRNA.To design a negative control siRNA, scramble the nucleotide sequence of the gene-specific siRNA and conduct a search to make sure it lacks homology to any other gene. A negative control siRNA with the same nucleotide composition as your siRNA but which lacks significant sequence homology to the genome.The editors of Nature Cell Biology have recommended several controls (2). Design appropriate controls.Ī complete siRNA experiment should include a number of controls to ensure the validity of the data. We suggest using BLAST, which can be found on the NCBI server at: 3. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with more than 16-17 contiguous base pairs of homology to other coding sequences.We have not seen any correlation between the position of target sites on the mRNA and siRNA potency. Since some regions of mRNA may be either highly structured or bound by regulatory proteins, we generally select siRNA target sites at different positions along the length of the gene sequence.Since a 4-6 nucleotide poly(T) tract acts as a termination signal for RNA pol III, avoid stretches of > 4 T's or A's in the target sequence when designing sequences to be expressed from an RNA pol III promoter.Ambion researchers find that siRNAs with 30-50% GC content are more active than those with a higher G/C content.Choose target sites from among the sequences identified in Step 1 based on the following guidelines: Research at Ambion has found that typically more than half of randomly designed siRNAs provide at least a 50% reduction in target mRNA levels and approximately 1 of 4 siRNAs provide a 75-95% reduction. If desired, you may modify this target site selection strategy to design siRNAs with other dinucleotide overhangs, but it is recommended that you avoid G residues in the overhang because of the potential for the siRNA to be cleaved by RNase at single-stranded G residues. In Elbashir's and subsequent publications, siRNAs with other 3' terminal dinucleotide overhangs have been shown to effectively induce RNAi. This is also compatible with using RNA pol III to transcribe hairpin siRNAs because RNA pol III terminates transcription at 4-6 nucleotide poly(T) tracts creating RNA molecules with a short poly(U) tail. (1) that siRNAs with 3' overhanging UU dinucleotides are the most effective. This strategy for choosing siRNA target sites is based on the observation by Elbashir et al. Record each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Find 21 nt sequences in the target mRNA that begin with an AA dinucleotide.īeginning with the AUG start codon of your transcript, scan for AA dinucleotide sequences. Corresponding siRNAs can then be chemically synthesized, created by in vitro transcription, or expressed from a vector or PCR product.ġ.
#Ape dna editor rna sequence software#
Online support for the software takes the form of a wiki page, the link to which is given on the ApE homepage.If you prefer to design your own siRNAs, you can choose siRNA target sites in a variety of different organisms based on the following guidelines. The virtual restriction digest feature provides users with predictive gel images showing the expected fragment sizes. Available for both PCs and Macs, ApE allows users to annotate DNA sequences with primer binding sites, coding regions, etc., and can display these features on graphic plasmid maps.
#Ape dna editor rna sequence free#
ApE (“A plasmid Editor”) is a free (yes, free!) downloadable DNA sequence editor program that offers comparable functionality to commercially available programs. Molecular biologists are always monkeying around with DNA-we amplify it, we cut it up, and we put the pieces back together.