A single-cloning-step procedure for the generation of RNAi plasmids producing long stem–loop RNA

RNA interference (RNAi), used as a tool, has revolutionized the studies of gene function. Long stem–loop dsRNA has been proven the most effective trigger for down-regulating target transcripts in RNAi-positive trypanosomatid parasites. Here we describe a protocol for constructing plasmids that produce long stem–loops by using a single cloning step. Inverted repeats are first obtained by self-ligation of PCR products that contain a randomized segment at one of their ends and then inserted in a plasmid vector. The random sequences create the loop (or “stuffer”) of the hairpin. This methodology was tested in Leishmania (Viannia) braziliensis to constitutively knock down the mRNAs for the well-studied paraflagellar rod protein 1 and 2 (PFR1 and PFR2) genes and revealed that mRNA cleavage products are unusually stable in these parasites. The protocol is suitable for any plasmid (for constitutive or inducible expression) and for any organism in which long stem–loops can be used to elicit RNAi.

Outline of the protocol for generating stuffer-containing inverted repeat plasmids.Figure optionsDownload high-quality image (112 K)Download as PowerPoint slideHighlights
► A simple protocol for generating plasmids with “stuffer”-containing inverted repeats.
► The constructed RNAi plasmids produce long stem–loop RNA.
► Requires a single cloning step, which makes it suitable for systematic RNAi plasmid libraries.
► Appropriate for any plasmid vector and any organism in which long dsRNA can be used to elicit RNAi.
► The methodology was tested for knocking down Leishmania (Viannia) braziliensis mRNAs.