standard Short vs. Long Hairpin RNAs for RNAi?

June-sun “Sunny” Yoon, is a graduate student at the University of Kentucky where he is working on the mechanisms and applications of RNAi. He is the author of an interesting paper recently highlighted in Technology Topics. MORE ABOUT THE AUTHOR

Bartoletti and colleagues recently published a paper in Genesis: The Journal of Genetics and Development that reports short hairpin RNAs being more effective than long hairpins in causing loss-of-function phenotypes of pointed (Pnt)  in Drosophila melanogaster.

RNA interference (RNAi) has been used widely in plants, insects, nematodes, and mammals to ‘silence’ (knock down) gene expression. It remains an important tool for conducting functional genomics studies but what the most effective approach to designing such studies remains a question of interest.

Structure of the Valium20 vector The upper drawing shows the layout of shRNA in the skeleton of the microRNA. The vector uses the vermilion + gene (orange) as a selective marker and possesses gypsy insulators (purple) on both sides. To control the expression level in the GAL4-UAS system, one of two 5xUASs (white) may be removed using the Cre/loxP method. The TRiP generated the VALIUM (Vermilion-AttB-Loxp-Intron-UAS-MCS) series of vectors for the incorporation of RNAi hairpins into attP landing sites (Ni et al. 2008, 2009, 2011). Go to the Drosophila Resource Center (DRSC) for more information about these vectors.

Bartoletti et al. targeted knockdown of the transcriptional activator, Pointed (Pnt), known to be downstream in the highly conserved Receptor Tyrosine Kinase (RTK) signaling pathway. Three RNAi Drosophila lines were generated in this study expressing hairpin RNA of Pnt; 1) Valium10 and 2) VDRC produce long hairpin RNAs and 3) Valium20 produces short hairpin RNAs (shRNA). The authors utilized the RNAi lines to assess loss of function of Pnt in several tissues and examined the phenotypic effects and efficacy of the lines.

Bartoletti et al. concluded that the Valium20 line producing shRNA was very effective in knocking down Pnt expression and producing distinct phenotypes in both embryonic and postembryonic developmental stages compared to the other RNAi lines expressing long hairpin RNAs.

Bartoletti et al. concluded

“As the Valium10 and VDRC RNAi lines produce phenotypes similar to that of a hypomorphic allele, these could be used in future to perform a genetic modifier screen while the Valium20 RNAi line that phenocopies a null allele could be used for performing RNA Sequencing experiments to identify genome wide transcriptional targets of Pnt.”


Interestingly, co-expression of Dicer2 in long hairpin RNA-producing Valium10 and VDRC  enhanced the severity of the phenotypes suggesting that Dicer2 improved RNAi efficiency significantly. Since Dicer2 is known to be a central player in processing long double-stranded RNAs into small interfering RNA, it seemed that the additional Dicer2 was needed to process the long hairpin RNAs in these lines.  With addition Dicer2 the long hairpin RNA-lines were as effective as the shRNA line.

The results of Bartoletti et al. are interesting and helpful but it is clear that the question of the most effective approach to RNAi experiments remains unresolved since Miller et al. 2012, for example, found that in Tribolium castaneum long dsRNAs (more than 300bp) and intermediate length dsRNAs (69bp) were efficient in knocking down gene expression in  while short dsRNA (31bp) were not.


Bartoletti R, Capozzoli B, Moore J, Moran J, Shrawder B, Vivekanand P. Short hairpin RNA is more effective than long hairpin RNA in eliciting pointed loss-of-function phenotypes in Drosophila. genesis. 2017;55:e23036.



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