• Custom siRNA Design Tool User Guide



    The siDESIGN Center is an advanced, user-friendly siRNA design tool which significantly improves the likelihood of identifying functional siRNA. One-of-a-kind options are available to enhance target specificity and adapt siRNA designs for more sophisticated experimental design.

    Note: Candidates produced using the siDESIGN Center do not carry the an siRNA product performance guarantee. However, siRNAs designed using this tool are more likely to provide potent and specific knockdown than those designed with conventional methods.


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    A. Identifier Type - Select Target Nucleotide Sequence

    There are four options for identifying the target sequence of interest for evaluation by the siDESIGN Center:

    1. Accession Number: The preferred accession numbers are found in the Reference Sequence (RefSeq) database, a non-redundant, curated, and annotated collection of sequence records for major model organisms. RefSeq accession numbers for mRNA begin with the characters NM_ or XM_, non-coding RNA accessions begin with NR_ or XR_, followed by a number of six or more digits.
        Examples:
      • NM_181838 (UBE2D2: ubiquitin-conjugating enzyme E2 D2)
      • NM_012165 (FBXW3: F-box and WD-40 domain protein 3)
      • NM_001083586 (CUGBP2: RNA binding protein 2)
      • NR_002323 (TUG-1; Taurine upregulated 1)

    2. Nucleotide Sequence: The Nucleotide box accepts FASTA-formatted sequence data up to 10,000 nucleotides. Only RNA or cDNA sequence should be entered. Sequences should be formatted using standard nucleic acid codes: A, C, G, T, and U. Any other alphanumeric entries will be ignored.
      • FASTA format requires a ">" symbol and a line break prior to entering or pasting the nucleotide sequence. It is recommended (but optional), that a Name be entered prior to the line break.
      • Use of Nucleotide Sequence as the Target Identifier will preclude the ability to select the Region (3' UTR, ORF, or 5' UTR) of the siRNA designs.

      • Example of FASTA format:
        FASTA format example

    3. Gene ID: A unique identifying number in the NCBI RefSeq database.
        Examples:
      • 672 (BRCA1)
      • 25 (ABL1)
      • 983 (CDC2)
      • 6419 (SETMAR)

    4. GI Number (GenInfo Identifier): A series of digits that are assigned consecutively to each sequence record processed by NCBI. The GI number bears no resemblance to the Accession number of the sequence record. If a sequence changes in any way, a new GI number will be assigned. Be sure the GI Number provided is for the nucleotide, not the protein sequence of interest.
        Example:
      • 27436944 (Homo sapiens lamin A/C (LMNA), transcript variant 2, mRNA)

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    B. Advanced Options: Designing Variant-specific siRNA

    This option supports isoform or gene variant-specific siRNA design. Please note that using this option may greatly reduce the number of siRNA designs generated due to the increased stringency of the design parameters.

    The Target Gene Variants table displays the Accession number(s) for all know RefSeq variants associated with the target gene (based on the Gene ID, GI number, or Accession number entered in Step 1). As a default, all variants will be targeted by the siRNA designs.

    • Click "Required" for any variant that must be targeted by the designs. Please note that at least one variant must remain "Required" for the design step to proceed.
    • Click "Allowed" for any variant that will neither be required nor excluded by resulting siRNA designs.
    • Click the "Excluded" radio button for any variant whose sequence record should NOT be targeted. Use of Advanced Options to exclude a variant will require selection of a BLAST database in Step 4.

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    C. Advanced Options: Additional Required Targets

    This option supports (a) the design of cross-species siRNA to target homologous genes in multiple organisms and (b) the design of siRNA to target conserved gene family members within an organism. Please note that using this option may greatly reduce the number of siRNA designs generated due to the increased stringency of the design parameters.

    Up to 10 Accession numbers may be entered (separated by spaces or line breaks) in the field provided. These gene targets will be "Required" by the design tool along with the original target gene provided in Step 1.


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    D. Region for siRNA Design

    Note: This option is not available if a Nucleotide Sequence is used as the Identifier in Step 1.

    • 5' UTR (Untranslated region) - Check this box to include the 5' UTR region for design consideration when the ORF offers a limited number of candidates because of sequence composition (high GC content) or if experimental strategies require it.
    • ORF (Open Reading Frame) (Default) - Recommended. This region is more highly conserved and less likely to be polymorphic. Non-coding RNAs will be treated as an ORF region. No designs will be returned if ORF is not selected as a target region when designing siRNAs against characterized non-coding RNAs.
    • 3' UTR (Untranslated region) - Check this box to include the 3' UTR region when the ORF offers a limited number of candidates because of sequence composition (high GC content) or if experimental strategies require it.

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    E. Indicate Organism - Seed Region Analysis

    This step is only required when Nucleotide Sequence is used as an Identifier in Step 1. The organism will be populated automatically from the RefSeq record if Gene ID, GI number, or Accession number is used.

    Select the corresponding organism from the drop-down list for human, mouse, or rat sequences. Any other organism should be set to "Other", and will not be analyzed for seed region frequency.

    Seed region analysis determines the number of exact matches of the siRNA seed region (nucleotides 2-7) to the 3'UTRs of all genes in the chosen genome (only performed for human, mouse, and rat). siRNAs with low seed region frequency will have reduced likelihood of seed-region mediated off-target effects compared to those with higher frequencies1 .

    This analysis will not limit the number of designs generated, but instead provides you with an indicator on the Results Page of the designs with low frequency of seed matches.

    1Anderson, E, et al., "Low Genomic 3' UTR Seed Complement Frequency Predicts siRNA Target Specificity". Poster at Keystone Symposia: MicroRNAs and siRNAs: Biological Functions and Mechanism. (January 2007)


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    F. GC content (%)

    This value is calculated using the core 19 nucleotide target sequence and does not include the dinucleotide leader sequence. Early research suggested that siRNA should have a G/C content of approximately 50%. For genes that are G/C poor or rich, the range of G/C content be expanded to include other ranges.

    • Minimum 30, Maximum 64 GC% (Default) - Recommended range

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    G. BLAST Options

    A BLAST step is required when using Advanced Options to exclude target gene variants.

    • No BLAST: Select this option if a BLAST database is unavailable through this tool for the desired species or if specificity is less of a concern compared to functionality.
    • Select BLAST Database: Select a species-specific database from the drop-down list of available databases to perform BLAST analysis on siRNA candidates.

    The following species-specific BLAST databases are available for analyses of candidate siRNAs: Human (Homo sapiens), Mouse (Mus musculus), Rat (Rattus norvegicus), Zebrafish (Danio rerio), Fruitfly (Drosophila melanogaster), Worm (Caenorhabditis elegans), Cow (Bos taurus), Rhesus monkey (Macaca mulatta), Chimpanzee (Pan troglodytes), Dog (Canis familiaris), and Chicken (Gallus gallus).

    Two combined databases are also available to support cross-species designs: Human/Mouse, and Human/Rat.

    If your species does not appear in this list, we recommend using a database most closely associated with the species of interest and/or performing an additional blastn analysis at NCBI with the candidate siRNAs.


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    H. RESULTS

    In addition to user-specified design criteria, candidate siRNA design regions are also selected for the required target(s) with other important criteria. Several of these key criteria include the exclusion of:

    • Motifs correlated with toxicity
    • Known miRNA seed region motifs
    • Known single nucleotide polymorphisms (SNPs)

    Following analysis, up to 50 of the top-scoring candidates are presented as a list. This page also includes details of the designated target for verification of the search.

    The output may be sorted by category by selecting the desired column heading:

    1. Selected (No sort function) - Click to choose siRNA designs that will be added to your shopping cart with default synthesis options. These options may be modified after siRNAs are added to the cart.
    2. Sense strand sequence - This represents the 19-nucleotide target. 3' -UU overhangs will be added when items are placed in the cart for synthesis
    3. Region -Region where the design aligns to the mRNA record. This field will indicate "N/A" if a Nucleotide sequence was provided as the Target Identifier.
    4. Start Position- Position of the siRNA design relative to the first base of the RNA record or Nucleotide sequence.
    5. GC% - GC% for the 19-base target.
    6. Score - Algorithmic indicator of the likelihood of successful silencing with this siRNA design. This score does not quantify expected percent knockdown.
    7. Low Seed Frequency- Yes/No indicator of a low siRNA seed region frequency within the 3'UTR of the target (or host) genome. siRNA with low frequency seed regions will have reduced off-target effects relative to higher frequencies1. This field will only be populated for Human, Mouse, or Rat targets.
    8. Min # mismatches (Antisense) - The lowest number of mismatches of the 19-base antisense strand to a sequence record other than the required target(s) as determined by BLAST analysis. Example - A score of "3" in this field would indicate that the antisense strand has no more than a 16/19 base match to any record in the BLAST database.
    9. Min # mismatches (Sense) - The lowest number of mismatches of the 19-base sense strand to a sequence record other than the required target(s) as determined by BLAST analysis. Example - A score of "3" in this field would indicate that the sense strand has no more than a 16/19 base match to any record in the BLAST database.
    10. Requires Sense Modification- Yes/No indicator of whether the siRNA design should be synthesized with a chemical modification to inhibit sense-strand uptake. This requirement is indicated when (a) The sense strand has fewer than 2 mismatches to a (non-self) sequence record in the BLAST database or (b) analysis indicates that the design favors sense strand over antisense strand entry into RISC. If "No BLAST" was selected, only criteria (b) will be applied.

    1Anderson, E, et al., "Low Genomic 3' UTR Seed Complement Frequency Predicts siRNA Target Specificity". Poster at Keystone Symposia: MicroRNAs and siRNAs: Biological Functions and Mechanism. (January 2007)


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