Procedure: 

1. Find the target gene sequence through PubMed under the category "Nucleotide". (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&itool=toolbar) 

2. Generate all possible secondary RNA structures of target sequences using software "Mfold". (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form) 

3. Click "ss-count file" on the output page of "Mfold" as shown below:

The opened ss-count file looks like this:

4. Copy all the content of the opened ss-count file and submit your job to our server. The H-b index of local regions of mRNA targeted by a siRNA will be calculated automatically.

5. Choose target regions that has a low H-b index value. 

6. Avoid those target regions that have high probability of forming a hairpin structure. 

7. Use BLAST to compare the potential target sites to the appropriate genome database (human, mouse, rat, etc). Eliminate those that have more than 16-17 contiguous base pairs of homology to other coding sequences. (http://www.ncbi.nlm.nih.gov/BLAST/)

Note: Your input to the Mfold server should be your target mRNA sequence but not a short siRNA sequence. For example: if you want to silence Bax mRNA, you input the Bax mRNA sequence into the Mfold server and you'll see the link for "ss-count file" of Bax mRNA. After copy the content of "ss-count file" of Bax mRNA into our H-b index server and submit the job, you'll know which region/sequence is best for silencing Bax mRNA.