1
Jan

2shRNA

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* Update 8-4-2010: separated forms for pLVTHM vector and pLKO.3G
* Update 3-11-2010: a modified version for pLKO, pQCtrc and other 21-mer vector is running at http://en.dogeno.us/2shRNAtrc

The original service "2-shRNA" runs on the unc server. I may lose the control of that site due to graduation and leaving. So I mirror the 2-shRNA oligo designer in my personal blog here. Enjoy!

2shRNA Oligo Designer for pLentilox, pLVTHM and others using 19-mer shRNA target

designer for pLentilox, using Blunt/XhoI insertion sites

target symbol:

  • siRNA sequence
  • 19 nt length check
  • U6 promoter requires G as the first nt
  • loop region
  • 5 end enzyme 5-|-3 (leave blank if it is blunt)
  • 3 end enzyme site 5-|-3 (sticky end represents as 5-C | TCGAG-3 for XhoI)
  • show Dharmacon 0-10 score calculation steps

designer for pLVTHM, using Mlu1/Cla1, with linker hack, no asymmetry hack

target symbol:

  • siRNA sequence
  • 19 nt length check
  • loop region
  • 5 end enzyme 5-|-3 (sticky end represents as 5-A | CGCGT-3 for Mlu1)
  • 3 end enzyme site 5-|-3 (sticky end represents as 5-AT | CGAT-3 for Cla1)


How to use 2shRNA?

  1. Use Dharmacon siDESIGN Center to find functional RNAi 19nt sequences (If the link is not working, please try google siDESIGN);
  2. Input the siRNA sequence here to get the stem-loop oligos;
  3. Order desalted oligos from Integrated DNA Technologies or other companys;
  4. Do "wet" experiments.

Useful Information

I. Criteria for identifying functional siRNA described by Reynolds and colleagues

  • from Rational siRNA design for RNA interference [Nature Biotechnology 2004 22: 326-330, PMID: 14758366]
  • low G/C content
  • a bias towards low internal stability at the sense strand 3'-terminus
  • lack of inverted repeats
  • sense strand base preferences (positions 3, 10, 13 and 19)

II. Algorithm of Dharmacon score (New Dharmacon score is ranging from 0-100, while here I still calculate 0-10 value. According to my experience, >4 is fine and higher may be the better.)

  1. 30%-52% GC content - Add 1 point for satisfying this criterion
  2. Three or more A/Us at positions 15-19 (sense) - Add 1 point for each A/U for a total up to 5 points. At least 3 points are required to be scored as positive ("+") in the final output
  3. The absence of internal repeats or hairpins as measured by a Tm < 20 degrees C. - Add 1 point for satisfying this criterion
  4. A at position 19 (sense) - Add 1 point for satisfying this criterion
  5. A at position 3 (sense) - Add 1 point for satisfying this criterion
  6. U at position 10 (sense) - Add 1 point for satisfying this criterion
  7. No G/C at position 19 (sense) - Subtract 1 point for not satisfying this criterion
  8. No G at position 13 (sense) - Subtract 1 point for not satisfying this criterion

III. Asymmetry for siRNA function

  • from Asymmetry in the Assembly of the RNAi Enzyme Complex [Cell 2003 115: 199-208, PMID: 14567917]

IV. Melting temperature predicted by Expanded Nearest-Neighbor Model

  • from Thermodynamic Parameters for an Expanded Nearest-Neighbor Model for Formation of RNA Duplexes with Watson-Crick Base Pairs [Biochemistry 1998 37: 14719-14735, PMID: 9778347]
  • Set a total strand concentration of 0.2 mM
  • RNA thermodynamic parameters in 1M NaCl, pH 7

* 20080526, source file: old html, perl. Enjoy!

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