RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics

Abstract

Background: The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA.

Results: We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery.

Conclusions: This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems.

Figure 1. Double-stranded RNA synthesis for RNA-interference

(A) Schematic representation of PCR products that can be used as templates for dsRNA synthesis. Oligonucleotide primers with sequences homologous to the multiple cloning site (MCS) can be used to amplify template for various gene constructs on the same vector backbone. Template for gene-specific or fragment-specific dsRNA synthesis can be synthesized using primers specific to the ends or specific regions withn the cDNA sequence, respectively. RNA polymerase promoter sequences present in the 5′ end of all primers (T7 is used as an example here) allow for dsRNA transcription using the PCR products as templates. (B) Argonaute-2 dsRNA prepared from a single transcription reaction to induce RNAi in planarians. DsRNA analyzed by non-denaturing 1% agarose gel electrophoresis with (+) or without (-) undergoing annealing, DNase I, or ethanol precipitation (EtOH ppt) treatments after transcription. (C) Single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) analyzed by nondenaturing 1% agarose gel electrophoresis. (D) Freshly and previously (2.5 years ago) synthesized dsRNA analyzed by non-denaturing 1% agarose gel electrophoresis. The position of ssRNA (*), dsRNA (**), and higher complexes (***) during electrophoresis is indicated, as are the positions of DNA size markers (left on (B)).

Figure 2. Ingestion and detection of dsRNA in planarians

(A) Illustration of in vitro synthesized dsRNA feeding. Liver puree carrier colored with food dye is mixed with a pestle. Centrifugation then isolates large liver matter to bottom of eppendorf tube and supernatant is aliquoted. DsRNA is added to aliquots by swirling with pipette tip. Food containing dsRNA is smeared into the bottom of the container housing planarians, which are then monitored for ingestion of dsRNA by coloration. (B-J) Ingested dsRNA is detected in the intestine and active in neoblasts within four days. Detection by fluorescent in situ hybridization (FISH) shows no ccdB control RNA (bacterial sequence) detected in unfed planarians (B), but present throughout the intestine of planarians fed ccdB dsRNA one day post-feeding (C). Detection of ccdB dsRNA is lost mostly (D) or entirely (E) four days post-feeding. (F) Smedwi-1 sense probe failed to detect antisense RNA in planarians fed germinal histone H4 dsRNA. Detection of Smedwi-1 expression in neoblasts by FISH (G) is reduced after dsRNA ingestion (H-I). Smedwi-1 dsRNA is detected in the intestine one day (H), but to a much lesser extent four days (I-J) post-ingestion. Asterisks represent planarian eye location. Scale bars = 0.5 mm.

Figure 3. Dynamics of RNAi activity after dsRNA ingestion in planarians

(A-R) Whole-mount in situ hybridization used to monitor Smedwi-1 (A-E), Smed-bruno-like (F-J), Bicaudal C (K-N), and germinal histone H4 (O-R) mRNA expression in asexual (A-J) or sexual (K-R) planarians subjected to a single dsRNA feeding. Times after feeding are indicated at the top of each panel. Fraction of animals representative of imaged sample is shown in parenthesis. Scale bars = 0.5 mm (A-J) and 1 mm (K-R).

Figure 4. Functional assessment of variations in dsRNA feeding methodology

(A-G) RNAi penetrance measured by monitoring survival of planarians subjected to a single feeding of liver paste containing 100 ng/μl of purified Ago-2 dsRNA (except panel E). Planarians fed with control dsRNA were unaffected (not shown). Daily time points after feeding are depicted along the x-axis. (A) Unpurified dsRNA transcription reaction (gray) compared to dsRNA treated with DNase, annealed and ethanol precipitated (black). (B) dsRNA stored frozen for over 2.5 years (gray) compared to recently synthesized dsRNA (black). (C) RNAi activity of liver paste preparation including agarose (gray) compared to dsRNA without agarose (black). (D) Ago-2 RNAi penetrance compared between larger (7-9 mm long; black) and smaller planarians (1-3 mm; gray). (E) Analysis of the effects of Ago-2 dsRNA concentration on RNAi penetrance. Death of planarians fed Ago-2 dsRNA at a concentration of 10 ng/μl (gray circles) compared to 100 ng/μl (black squares) and 1000 ng/μl (gray triangles) concentration. (F) Effects of multiple dsRNA feedings on Ago-2 RNAi phenotype. Planarians fed once with Ago-2 dsRNA (gray squares) compared to those fed twice (black circles) or three times (gray triangles). (G) Effects of multiple feedings compared to multiple Ago-2 dsRNA feedings on appearance of phenotype. Death of planarians fed Ago-2 dsRNA once (gray squares), were compared to those fed Ago-2 dsRNA twice (gray circles), and to planarians fed once with Ago-2 dsRNA and once with control dsRNA (black circle). (H) Analysis of correlation between P2X-A dsRNA concentration and increased fission induced by P2X-A RNAi in D. japonica. Number of fission events per group (x-axis) after last of three feedings with increasing concentrations (see figure) of control (left) or P2X-A (right) dsRNA. Timing of fission events after the third of three feedings is shown on y-axis. n ≥ 10 for each category.

Figure 5. Phenotypes manifest more rapidly in animals fed dsRNA synthesized in vitro compared to E. coli-expressed dsRNA

(A) Range of lysis phenotypes observed after nkx2.2 knockdown, increasing in severity from left (early/no phenotype) to right (late/complete lysis and death). Phenotypes manifested earlier and progressed more quickly for animals fed in vitro-synthesized nkx2.2 dsRNA. (B) Plot of the percentage of animals (Y-axis) that lyse (dashed lines) or survive (solid lines) for the number of days indicated (X-axis) after a single feeding of either E. coli expressing nkx2.2 dsRNA (black diamonds) or in vitro-synthesized nkx2.2 dsRNA at 20, 40, and 80 ng/μl (gray, yellow and red circles, respectively). n ≥ 24 for each feeding category. Scale bars = 1 mm.