shRNA

 

shRNA (short hairpin RNA) is a DNA molecule that can be cloned into expression vectors to express siRNA (19-21nt RNA duplex) for RNAi interference studies. shRNA has the following structural features:

               

·         A short nucleotide sequence ranging from 19-29 nucleotides derived from the target gene, followed by

·         A short spacer of 4-15 nucleotides (i.e. loop) and

·         A 19-29 nucleotide sequence that is the reverse complement of the initial target sequence

 

Invitrogen offers shRNA design (BLOCK-iT™ RNAi Designer) and synthesis services (Custom Primer Resources) and vectors for shRNA expression. Use BLOCK-iT(TM) Inducible H1 RNAi Entry Vector Kit for inducible shRNA expression and BLOCK-iT™ U6 Entry Vector kit for constitutive expression.

 

 

shRNA Design

 

The BLOCK-iT™ RNAi Designer finds 21 nt gene-specific target sequences that fulfill proprietary design criteria and reports up to 10 top scoring target sequences.

 

For the selected target sequences, the designer:

·         Uses selected default or entered custom loop sequence

·         Uses appropriate linker sequences for directional cloning into Invitrogen's BLOCK-iT^TM entry vector (pENTR^TM/U6) vector or BLOCK-iT™ H1/TO vector

·         Designs top and bottom strands of the shRNA oligo, incorporating linker, target, loop and reverse complement of the target sequence.

·         Reports top strand, bottom strand and the annealed oligo sequence

 

See Online Help for additional information.

 

shRNA Ordering

 

Order both top and bottom DNA strands from Invitrogen Custom Primer Services and anneal both strands to generate a double-stranded oligo with 4-nucleotide overhangs suitable for directional cloning into Invitrogen's vectors. Check shRNA annealing protocol for annealing the ss DNA oligo.