Of interst: Chen et al. "Transcript level is a key factor affecting RNAi efficiency"

Pestic Biochem Physiol. 2021 Jul;176:104872. doi: 10.1016/j.pestbp.2021.104872.

Transcript level is a key factor affecting RNAi efficiency.

Chen J, Peng Y, Zhang H, Wang K, Tang Y, Gao J, Zhao C, Zhu G, Palli SR, Han Z

Abstract:

Efficiency is the basis for the application of RNA interference (RNAi) technology. Actually, RNAi efficiency varies greatly among insect species, tissues and genes. Previous efforts have revealed the mechanisms for variation among insect species and tissues. Here, we investigated the reason for variable  efficiency among the target genes in the same insect. First, we tested the genes sampled randomly from Tribolium castaneum, Locusta migratoria and Drosophila S2 cells for both their expression levels and sensitivity to RNAi. The results indicated that the genes with higher expression levels were more sensitive to RNAi. Statistical analysis showed that the correlation coefficients between transcript levels and knockdown efficiencies were 0.8036 (n = 90), 0.7255 (n = 18) and 0.9505 (n = 13), respectively in T. castaneum, L. migratoria and Drosophila S2 cells. Subsequently, ten genes with varied expression level in
different tissues (midgut and carcass without midgut) of T. castaneum were tested. The results indicated that the higher knockdown efficiency was always obtained in the tissue where the target gene expressed higher. In addition, three genes were tested in different developmental stages, larvae and pupae of T. castaneum. The results found that when the expression level increased after insect pupation, these genes became more sensitive to RNAi. Thus, all the proofs support unanimously that transcript level is a key factor affecting RNAi sensitivity. This finding allows for a better understanding of the RNAi efficiency variation and lead to effective or efficient use of RNAi technology.

DOI: 10.1016/j.pestbp.2021.104872
PMID: 34119217 [Indexed for MEDLINE]

Video report -- cell-based assay

Peters KA, Detmar E, Sepulveda L, Del Valle C, Valsquier R, Ritz A, Rogers SL, Applewhite DA. A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells. J Vis Exp. 2018 Aug 19;(138). doi: 10.3791/58325. PubMed PMID: 30176023.

FlyBook review on RNAi screening in Drosophila cells and in vivo

Heigwer F, Port F, Boutros M. RNA Interference (RNAi) Screening in Drosophila. Genetics. 2018 Mar;208(3):853-874. PMID: 29487145.

From the abstract: "... RNA interference (RNAi) ... has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila, RNAi has been applied in cultured cells or in vivo to perturb the function of single genes or to systematically probe gene function on a genome-wide scale. In this review, we will describe the use of RNAi to study gene function in Drosophila with a particular focus on high-throughput screening methods applied in cultured cells. ..."

New report on qPCR design for RNAi knockdown effectiveness testing

Mainland RL, Lyons TA, Ruth MM, Kramer JM. Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines. BMC Res Notes. 2017 Nov 29;10(1):647. PMID: 29187229.

From the abstract: "... the strongest gene knockdown was detected when qPCR was performed using 5' primer sets in combination with mRNA-derived cDNA. Our results indicate that detection of undegraded mRNA cleavage fragments can result in underestimation of true knockdown efficiency for a RNAi construct. ..."

Short vs. long hairpins in fly RNAi

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 Jul;55(7). PMID: 28464429.

From the abstract: "Pointed (Pnt) is a transcriptional activator ... As mutations in pnt perturb the development of several tissues, we wanted to examine the effect and efficacy of using RNAi to target Pnt. We have expressed pnt RNAi in the eyes, oocyte, and heart cells using three different RNAi lines: Valium20, Valium10, and VDRC. Valium20 is distinct since it generates a short hairpin RNA (shRNA), while Valium10 and VDRC produce long hairpin dsRNA. We found that for each tissue examined Valium20 exhibited the strongest phenotype while the Valium10 and VDRC lines produced varying levels of severity and that the long hairpin RNA produced by the Valium10 and VDRC lines are unable to effectively knockdown pnt in embryonic tissues."

Methods publications relevant to cell and in vivo RNAi

Methods in Molecular Biology has recently published papers relevant to Drosophila cell culture, cell-based RNAi, and in vivo RNAi.

Debec A, Megraw TL, Guichet A. Methods to Establish Drosophila Cell Lines. Methods Mol Biol. 2016;1478:333-351. PubMed PMID: 27730593. 

Billmann M, Boutros M. Methods for High-Throughput RNAi Screening in Drosophila Cells. Methods Mol Biol. 2016;1478:95-116. PubMed PMID: 27730577.

Kaya-Çopur A, Schnorrer F. A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila. Methods Mol Biol. 2016;1478:117-143. PubMed PMID: 27730578.

Methods paper -- in vivo RNAi screens

Zhou J, Tong C. Design and Methods of Large-Scale RNA Interference Screens in Drosophila. Methods Mol Biol. 2016;1470:163-9. PMID: 27581292.

From the abstract: "... Here, we discuss methods for the design and performance of a large-scale in vivo RNAi screen in Drosophila. Furthermore, methods for the validation of results and analysis of data will be introduced."

Using the white gene as an indicator of CRISPR-based editing

Ge DT, Tipping C, Brodsky MH, Zamore PD. Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Co-Conversion. G3 (Bethesda). 2016 Aug 19. pii: g3.116.032557. PMID: 27543296.

From the abstract: "... Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. ... We find that end joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy."

Talk of transvection, RNAi, and Gal4-UAS, all in one G3 report

Noble et al. in G3: "important new implications for experimental design utilizing the GAL4-UAS system" https://t.co/v7ArcAR4vU

— Stephanie Mohr (@smohrfly) August 5, 2016

Persistence of RNAi in mosaic studies

Another report regarding in vivo RNAi that fly folks using the technique should be aware of (see this post for another, and check out other posts tagged "in vivo RNAi" or "troubleshooting").

Bosch JA, Sumabat TM, Hariharan IK. Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis yet Enables Highly Sensitive Lineage Tracing. Genetics. 2016 Mar 16. PMID: 26984059.

From the abstract: "... By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. ... knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked "shadow RNAi" clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased ... Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. ..."

Another paper reporting concern about an RNAi fly library

Any of us using the relevant RNAi stocks should be aware.

Vissers JH, Manning SA, Kulkarni A, Harvey KF. A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth. Nat Commun. 2016 Jan 13;7:10368. PMID: 26758424.
From the abstract: "... Here we investigate an important technical limitation with the widely used VDRC KK RNAi collection. We find that approximately 25% of VDRC KK RNAi lines cause false-positive enhancement of the Hippo pathway, owing to ectopic expression of the Tiptop transcription factor. Of relevance to the broader Drosophila community, ectopic tiptop (tio) expression can also cause organ malformations and mask phenotypes such as organ overgrowth. To enhance the use of the VDRC KK RNAi library, we have generated a D. melanogaster strain that will allow researchers to test, in a single cross, whether their genetic screen of interest will be affected by ectopic tio expression."

Drosophila cell team at the DGRC reports integrase-mediated cassette exchange approach to production of clonal transgenic fly cell lines

Lucy Cherbas1, Jennifer Hackney, Lei Gong, Claire Salzer, Eric Mauser, Dayu Zhang and Peter Cherbas. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines.
Early online in G3.

From the abstract: "We describe an adaptation of ΦC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking site lines carries a single mapped copy of one of the docking platforms. ... We describe procedures for isolating cells carrying the substitutions ... When compared with clonal lines made by traditional transformation methods ... targeted insertion lines give more uniform expression, lower basal expression and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays – a major emphasis of cell-based studies." 

Protocol paper -- qPCR to detect cell death-related transcripts following dsRNA treatment of fly cells

Denton D, Kumar S. Analyzing the Response of RNAi-Treated Drosophila Cells to Death Stimuli by Quantitative Real-Time Polymerase Chain Reaction. Cold Spring Harb Protoc. 2015 Jul 1;2015(7):pdb.prot086223. PMID: 26134907.

From the abstract: "A useful complement to animal studies is the use of Drosophila cell lines to analyze cell-death responses. ... Drosophila cell lines are very amenable to knockdown studies ... the cell lines are useful for investigating the response to death stimuli, following gene knockdown, by examining the expression of cell-death genes. This protocol describes the synthesis of dsRNA for treatment of Drosophila cells and the subsequent analysis of cell-death gene expression by quantitative real-time polymerase chain reaction (qPCR)."

CRISPR off-targets study in human cells

Findings relevant to fly studies? Will be interesting to see.

Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, Hwang J, Kim JI, Kim JS. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015 Mar;12(3):237-43. PMID: 25664545.

From the abstract: "... genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. ... We also showed that Cas9 nucleases can be highly specific ... and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. ..."

Sleep screens--methods review includes discussion of in vivo RNAi

Axelrod S, Saez L, Young MW. Studying circadian rhythm and sleep using genetic screens in Drosophila. Methods Enzymol. 2015;551:3-27. PMID: 25662449.

From the abstract: "... In this chapter, we briefly recall the history of circadian rhythm and sleep screens and then ... describe techniques ... for ... screening in the field.  ... comparing the newer approaches of transgenic RNA interference (RNAi) to classical forms of mutagenesis ... We discuss the different screening approaches in light of the literature and published and unpublished sleep and rhythm screens utilizing ethyl methanesulfonate mutagenesis and transgenic RNAi from our lab."

Anti-Cas9 antibody

If an anti-Cas9 antibody would be useful to your CRISPR-related projects--at least one company now reports availability of an anti-Cas9 monoclonal.

New report--CRISPR-Cas9 nickase system in Drosophila

Ren X, Yang Z, Mao D, Chang Z, Qiao HH, Wang X, Sun J, Hu Q, Cui Y, Liu LP, Ji JY, Xu J, Ni JQ. Performance of the Cas9 Nickase System in Drosophila melanogaster. G3 (Bethesda). 2014 Aug 15. pii: g3.114.013821. PMID: 25128437.

Study explores culture media for Drosophila cells

Burnette M, Brito-Robinson T, Li J, Zartman J. An inverse small molecule screen to design a chemically defined medium supporting long-term growth of Drosophila cell lines. Mol Biosyst. 2014 Aug 6. PMID: 25096480.

From the abstract: "Drosophila cell culture is used as a model system with multiple applications ...  To characterize the minimal requirements for long-term maintenance of Drosophila cell lines, we developed an inverse screening strategy to identify small molecules and synergies stimulating proliferation in a chemically defined medium. ... Validated factors were investigated for their ability to maintain cell growth over multiple passages in the chemically defined medium (CDM). The polyamine spermidine proved to be the critical component that enables the CDM to support long-term maintenance of Cl.8 cells. Spermidine supplementation upregulates DNA synthesis for Cl.8 and S2 cells and increases MAPK signaling for Cl.8 cells. The CDM also supports the long-term growth of Kc167 cells. Our target scoring approach validated the importance of polyamines ... Future iterations of the screen will enable the identification of compound combinations optimized for specific applications ... thus increasing the versatility of Drosophila cell culture as both a genetic and biochemical model system. ..."

Luciferase-based assays in cultured cells

Yun C, Dasgupta R. Luciferase reporter assay in Drosophila and Mammalian tissue culture cells. Curr Protoc Chem Biol. 2014 Mar 14;6(1):7-23. PMID: 24652620.

DRSC announces launch of FlyPrimerBank online resource

The DRSC is pleased to announce the launch of FlyPrimerBank.

FlyPrimerBank is built around a database of three precomputed qPCR primer pairs per Drosophila gene. Online search output includes information about the primer pairs and when available, validation test results from our group or the community at large.

For details please see our paper, just out in the journal G3. You can also check out the documentation page.

Important for RNAi studies, FlyPrimerBank can report overlap between the primers and RNAi reagents in public collections. This helps you avoid choosing primers that could amplify the reagent.

As always, your feedback on the online resource is welcome.

FlyPrimerBank also makes it possible for you to indicate which qPCR primers in FlyPrimerBank have or have not worked for them, or to upload the sequences of good primer pairs not already in the database.

With your help, the utility of the resource will further increase over time.