1. Sequence submitted - siRNA directed against any mRNA regions (UTRs or coding sequence) are renowned to be functionnal, but we default to and recommend using only the coding sequence (from ATG to stop codon) for better efficacity.

2. siRNA size - siRNAs ranging from 19 to 29 nt have been shown to effectively and specifically reduce the expression level of target mRNA. However, we recommend 21-nt siRNA by default because this length of siRNA can be more easily designed into various vectors, including miRNA-based shRNA.

3. Starting nucleotide - The first nucleotide of siRNA should be either adenine (A) or guanine (G) to ensure effective transcription by RNA Polymerase III. It is crucial to avoid using pyrimidines (cytosine - C or thymine - T) as the first nucleotide, as they may hinder proper recognition and binding by the RNA Polymerase III promoter. Thus, duplexes with a C or T at the 5' end are discarded.

4. Promoter - The U6 promoter shows a preference for guanine (G) as the starting nucleotide, while the H1 promoter exhibits a preference for adenine (A) as the starting nucleotide.

5. Thermodynamic properties - The 3' end of the sense strand should have lower thermodynamic stability compared to the 5' end. This design enhances siRNA functionality and target specificity by facilitating efficient target mRNA recognition and binding in the seed region. Thus, duplexes with a G or C at the 3' end are discarded.

6. GC content - It is generally recommended to choose sequences with a low GC content. This program defaults to using a GC content range between 30 % and 55 %.

7. Internal repeats - siRNA lacking internal repeats is strongly preferred. Sequences containing these internal repeats are discarded.

8. Immune response - Sequences known to induce immune response are discared.

9. Palindromes - siRNA lacking palindromes is strongly preferred. Candidates containing palindromes with a length of 8 or more bases are discarded.

10. BLAST against mRNA database - Both mature 5P and 3P are subjected to blasting to minimize off-target effects.

11. BLAST against miRNA databases - siRNA matches with highly conserved miRNA SEED regions will result in lower scores.

12. Loop sequence - RNABASE presets 25 loops sequences, ranging in length from 4 to 10 base pairs. By default, RNABASE uses "TCAAGAG" as the loop, but users can also specify a custom loop sequence.

13. Scoring system - The following criteria get scores: 1) Asymmetrical base pairing in the duplex; 2) Presence of "A" at the 6th, "T" at the 10th nucleotides in the antisense strand; 3) Absence of "T" at the 1st, "G" at the 19th nucleotides in the antisense strand; 4) More A/T in the first 7 bases of the antisense strand; 5) Energy valley from 9th to 14th nucleotides in the antisense strand; 6) Presence of "A" at the 3rd, "T" at the 10th, and "A" at the 19th nucleotides in the sense strand; 7) Absence of "G" at the 13th, "G/C" at the 19th nucleotides in the sense strand; 8) More A/T from 13th to 19th nucleotides of the sense strand;

14. Scores - The maximum overall score will be 15, and siRNAs with scores >= 6 can be considered as good candidates.

15. Order of sense and antisense - The popular practice is promoter-sense-loop-antisense. However, based on the principles of Dicer cleavage of precursor miRNA, the first two nucleotides at the 5' end of the antisense will be removed, and extra 'UU' at 3' end will be part of antisense. This means that the mature 3P sequence will not completely complement the target mRNA. To a large extent, mismatches at the 3' end do not significantly affect the functionality of the antisense but may reduce its binding affinity with the target mRNA. In light of this, we always recommend using a safer placement order, which is promoter-antisense-loop-sense. At the same time, we also retain the popular placement approach which you can find in the report.