pLV-RNAi Protocols – Design shRNA

Designing oligonucleotide encoding shRNA (sense-loop-antisense) sequence.

The features of single-strand oligonucleotide encoding shRNA sequence include the following (Fig. 3):
1. Stem-loop sequence. Consist of 19-21 nucleotides (nt) target (or sense) sequence, 10 nt loop sequence (TTGGATCCAA), and 19-21 nt target antisense sequence. This sense-loop-antisense sequence is a perfect palindrome with 48-52 nt in length. Therefore, only one oligonucleotide has to be ordered since both strands in the annealed double-strand oligonucleotide are identical (Advantages of the Product Fig. 2A

2. Overhang sequence. The 5’-AAAA sequence is complementary to the overhang of the pRNAi vectors and restores the promoter and terminator sequences in the vector (Advantages of the Product Fig. 2B).
There are no standard but only general guidelines for selecting the shRNA targeting sequences [1-5]. However, following these guidelines does not guarantee efficient knockdown. The effectiveness of shRNA sequence has to be determined by gene suppression analysis. In general, we suggest selecting 3 to 5 target sequences per gene of interest to identify at least two functional shRNAs with over 70% knockdown efficiency in target gene expression.

Listed below are a few rules we recommend for selecting shRNA sequences:

• Unique 19-21 nt target sequence with 30%-70% GC content; A 35%-55% GC content is preferred.
• Avoid target sequences with significant homology to other genes, unless the shRNA is intended to knockdown a gene family.
• A target sequence within an open reading frame is preferred; targeting 3’ UTR is ok.
• No more than 4 consecutive T in the shRNA sequence to cause early transcription termination.
• GC-rich in the 5’ end and AT-rich in the 3’ end of target sequences are preferred.
• Choose a target sequence that starts with guanosine (G) if the U6 promoter is used.

Figure 1. Structure of oligonucleotide encoding shRNA sequence.

Note: Please make sure the 5’ overhang sequence is AAAA and the loop sequence is TTGGATCCAA.
Suggestion: Ordering oligonucleotide with gel purification and 5´ phosphorylation is not required.

As a negative control for knockdown analysis, use a nonspecific target sequence or a scrambled sequence. For example, shRNA oligonucleotide targeting luciferase or ß-galactosidase gene could be used as negative control.

References:

1. Schwarz, D.S., et al., Asymmetry in the assembly of the RNAi enzyme complex. Cell, 2003. 115(2): p. 199-208.
2. Khvorova, A., A. Reynolds, and S.D. Jayasena, Functional siRNAs and miRNAs exhibit strand bias. Cell, 2003. 115(2): p. 209-16.
3. Reynolds, A., et al., Rational siRNA design for RNA interference. Nat Biotechnol, 2004. 22(3): p. 326-30.
4. Pei, Y. and T. Tuschl, On the art of identifying effective and specific siRNAs. Nat Methods, 2006. 3(9): p. 670-6.
5. Mittal, V., Improving the efficiency of RNA interference in mammals. Nat Rev Genet, 2004. 5(5): p. 355-65.