The synthesis of dsRNA in vitro and direct delivery by feeding mixed with planarian food as a carrier has proven successful in assaying gene function in a few recent studies (Collins et al., 2010; Pellettieri et al., 2010; Rouhana et al., 2010, 2012; Nakagawa et al., 2012; Sakurai et al., 2012). We exploited one advantage that in vitro dsRNA synthesis has over bacterial expression of dsRNA: it does not require subcloning genes or gene-fragments into a particular vector. Templates for dsRNA synthesis are generated by polymerase chain reaction (PCR) using primers that contain a specific RNA polymerase promoter sequence on the 5′-end and sequence corresponding to the gene of interest on the 3′-end (Fig. 1A). RNA molecules are synthesized simultaneously from the resulting templates in sense and antisense orientations, and are annealed during progression of the transcription reaction (see Experimental Procedures). The quality of dsRNA can be assessed by nondenaturing agarose gel electrophoresis. Double-stranded RNA appears as one or two intense bands prolonged as an upward smear that is not seen in DNA or single-stranded RNA (ssRNA) (Fig. 1B,C). The dsRNA migrates approximately (although not exactly) as predicted for DNA, and slower than ssRNA (Fig. 1C). It is not necessary to apply DNase to the dsRNA transcription reaction, denature and anneal the RNA molecules after transcription, or purify the synthesized product. The transcription reaction can be directly fed to planarians to induce RNAi, or stored at −20°C for long periods of time. We have observed that dsRNA is extremely stable and functional after being stored at −20°C for over 2 years (Fig. 1D; below).
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 transcription can be synthesized using primers that anneal to the ends or internal regions of 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 nondenaturing 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 nondenaturing 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)).
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The approach described for synthesis of dsRNA is also amenable if one desires to target different ends of a transcript or specific exons, as primers can be designed to target any particular region of a gene. Alternatively, template for dsRNA synthesis to target an array of different genes can be generated from a single primer pair that anneals to the vector sequence flanking the multiple cloning site of specific vectors (Fig. 1A). We have taken this approach to target sequences inserted in a common vector (pBluescript SK+) from a cDNA library (Zayas et al., 2005), in 96-well plate format.
Functional Assessment of Variables During RNAi-Mediated Silencing by Feeding In Vitro-Synthesized dsRNA
We wished to verify the robustness of RNAi-mediated silencing on gene function using our protocol. To do this, we targeted Argonaute-2 (Ago-2), a gene with conserved functions in micro-RNA and siRNA regulation of gene expression (reviewed by Kim et al., 2009). Ago-2 is broadly expressed in planarians, but previous studies have shown that RNAi-mediated silencing of this gene leads to gradual loss of neoblasts and ultimately death (Rouhana et al., 2010; Li et al., 2011). Thus, using Ago-2 RNAi-induced death as a readout for RNAi penetrance, we tested the effects of using different degrees of dsRNA purity, age, concentration, and inclusion of agarose in the carrier (Fig. 4A–C). We also tested variations in animal size, dsRNA concentration, and application of multiple dsRNA feedings, on RNAi-mediated gene silencing (Fig. 4D–H).
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 where noted in E-G). 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 with dsRNA treated with DNase, annealed and ethanol precipitated (black). B: dsRNA stored frozen for over 2.5 years (gray) compared with recently synthesized dsRNA (black). C: RNAi activity of liver paste preparation including agarose (gray) compared with 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 with 100 ng/μl (black squares) and 1,000 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 with those fed twice (black circles) or three times (gray triangles). G: Effects of multiple feedings compared with multiple Ago-2 dsRNA feedings on appearance of phenotype. Death of planarians fed Ago-2 dsRNA once (gray squares), were compared with 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 the last of three feedings with different 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.
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First, we tested whether RNAi by feeding planarians with purified dsRNA is more effective than feeding with unprocessed dsRNA transcription reaction (see Experimental Procedures). We did not find a noticeable difference in penetrance of Ago-2 RNAi by feeding pure Ago-2 dsRNA or unprocessed dsRNA transcription reaction (Fig. 4A). Next, we tested whether storage of dsRNA at −20°C for long periods of time affects RNAi activity. We found that feeding planarians dsRNA that had been frozen for 2.5 years (Fig. 1D) was just as effective in disrupting Ago-2 function as freshly synthesized dsRNA (Fig. 4B). Finally, we tested whether addition of agarose to the dsRNA/liver mixture, which facilitates handling during the feeding procedure, had an effect on RNAi penetrance. Previous experiments using bacterially expressed dsRNA revealed an improvement in RNAi penetrance by using dsRNA/liver paste (a.k.a. “SOFT SERVE”) over inclusion of agarose (Gurley et al., 2008). We failed to observe an effect on RNAi penetrance by inclusion of agarose, when using in vitro-synthesized dsRNA (Fig. 4C). However, planarians do not seem to retain as much food when agarose is present.
One caveat of feeding bacterially expressed dsRNA, as compared to dsRNA delivery by injection, is the uncertainty in amount of delivered nucleic acid. Feeding dsRNA synthesized in vitro allows for controlled concentration of nucleic acid in the food carrier. The amount of food that is ultimately ingested by planarians may vary. However, we assumed that the amount of food ingested would correlate with planarian size; therefore, keeping the concentration of dsRNA in the food constant would suffice to see similar RNAi penetrance in animals of various sizes. To test this, we subjected small (1–3 mm) and large (7–9 mm) planarians to a single feeding with excess amount of liver paste containing a constant concentration of Ago-2 dsRNA (100 ng/μl). Contrary to our expectations, we observed 75% survival of large animals subjected to Ago-2 RNAi days after all small animals had died (Fig. 4D). Next, we tested whether exposing small planarians to different concentrations of dsRNA could result in various degrees of Ago-2 RNAi penetrance. Small planarians were subjected to a single feeding containing 10 ng, 100 ng, or 1,000 ng of Ago-2 dsRNA per microliter of liver paste, and scored for survival. Animals presented with the smallest dose of dsRNA were mostly unaffected by the treatment up to 32 days postfeeding (Fig. 4E). Animals presented with the largest dose of dsRNA showed similar Ago-2 RNAi penetrance as those treated with the usual (100 ng/μl) dose of dsRNA (Fig. 4E). These results demonstrate that full manifestation of the Ago-2 RNAi phenotype is dose dependent and is achieved at approximately 100 ng/μl. We currently use this concentration for disrupting gene function by RNAi in individual experiments and screens. However, weekly feedings are routinely applied to induce robust RNAi in larger animals, including sexually mature animals (Collins et al., 2010; Chong et al., in revision) and/or to analyze phenotypes that require prolonged knockdown schemes (Rouhana et al., 2012; Roberts-Galbraith and Newmark, 2013).
Whether using dsRNA injection or feeding of bacterial or in vitro–synthesized dsRNA, researchers normally use multiple rounds of delivery to induce RNAi in planarians. To directly test whether multiple rounds of dsRNA feeding affect RNAi penetrance, planarians were fed Ago-2 dsRNA once, twice or three times, with 2 days separating each round of feeding. We observed that in fact, planarians subjected to two or three rounds of Ago-2 dsRNA feedings died approximately twice as fast as those subjected to only one round of feeding (Fig. 4F). Planarians subjected to one round of Ago-2 dsRNA feeding, died 20 to 28 days after the initial feeding (Fig. 4F). Whereas, planarians subjected to two rounds of dsRNA feeding died between 14 and 17 days after the initial feeding, and those subjected to three rounds died between 13 and 15 days after the initial feeding (Fig. 4F). These results suggested that multiple ingestions of dsRNA accelerate full manifestation of RNAi phenotype. However, it remained unclear whether multiple rounds of dsRNA delivery accelerated the manifestation of the Ago-2 RNAi phenotype due to more effective destruction of Ago-2 transcripts, or due to the demands of cell proliferation induced in response to feeding (Baguñà, 1976). To distinguish between these two scenarios, we first subjected planarians to an initial round of Ago-2 dsRNA feeding and a second feeding 3 days later with either control dsRNA or Ago-2 dsRNA. Planarians subjected to one round of Ago-2 dsRNA died 15 to 27 days after the initial feeding (Fig. 4G). Planarians fed Ago-2 dsRNA first and control dsRNA second, died between 14 and 20 days after the initial feeding (Fig. 4G). Animals subjected to two rounds of Ago-2 dsRNA died between 11 and 15 days after the initial feeding (Fig. 4F). These results show that multiple rounds of dsRNA feeding can enhance RNAi penetrance, although feeding alone causes a modest acceleration of the Ago-2 RNAi phenotype, likely due to a higher demand for neoblasts capable of proliferation after feeding.
Altogether, these results demonstrate the importance of controlling the amount of dsRNA delivered to planarians. Multiple rounds of dsRNA feeding can be used to enhance RNAi penetrance or accelerate phenotype manifestation. Within limits, increasing amounts of dsRNA can also improve phenotype manifestation. Experiments analyzing a neoblast receptor required to regulate nutritional influence on proliferation, P2X-A, support this observation (Fig. 4H). P2X-A RNAi leads to increased fission events in D. japonica (Sakurai et al., 2012). We found that increasing the concentration of P2X-A dsRNA had a limited correlation with the number of fission events observed after RNAi (Fig. 4H).
The concentration of dsRNA that most efficiently revealed Ago-2 or P2X-A phenotypes in our experiments, ranged between 10 and 100 ng/μl (Fig. 4E,H), but how do these amounts compare with RNAi protocols using bacterial expression of dsRNA? To directly analyze this, we silenced Smed-nkx2.2 expression by feeding planarians dsRNA produced in vitro or in bacteria. Smed-nkx2.2 is an intestinally expressed homeobox transcription factor required for neoblast proliferation (Forsthoefel et al., 2012). Knockdown of nkx2.2 initially causes lesioning on the dorsal surface of planarians in a pattern over the intestinal branches, which is followed by complete lysis and death (Fig. 5A). In animals fed in vitro-synthesized dsRNA, lysis begins sooner and progresses more rapidly than those fed E. coli-expressing nkx2.2 dsRNA (Fig. 5B). Planarians fed liver paste with concentrations of in vitro-synthesized nkx2.2 dsRNA of 20, 40 or 80 ng/μl showed lysis 8–10 days postfeeding, whereas planarians fed with E. coli showed lysis 9–13 days postfeeding (Fig. 5B). Death of planarians subjected to nkx2.2 dsRNA synthesized in vitro occurred 9–12 days postfeeding, whereas bacteria-fed counterparts died between 11 and 16 days postfeeding (Fig. 5B). Although it is possible that in some situations RNAi by bacterial feeding could be as effective as this protocol, these results demonstrate that RNAi using dsRNA synthesized in vitro leads to an earlier manifestation of nkx2.2 phenotypes than bacterially expressed dsRNA. Theoretically, such delays in phenotypic manifestation could make a difference between erroneous “false-negative” classification and functional revelation in direct experiments or screens.
Figure 5. Phenotypes manifest more rapidly in animals fed dsRNA synthesized in vitro compared with 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.
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In conclusion, feeding of in vitro–synthesized dsRNA is an effective alternative to more commonly used approaches for inducing RNAi in planarians. This simple approach is robust, noninvasive, and allows for direct assessment of dsRNA quantity and quality. Additional advantages of our methodology, which make it amenable for high-throughput screening, include low cost, labor and time requirements (Table 1). The feeding of in vitro–synthesized dsRNA is the most convenient way to inhibit gene function in planarians. Application of this protocol will accelerate efforts of researchers using the planarian as a model system in molecular, regenerative, and developmental biology research. Additionally, researchers trying to establish protocols for RNAi by dsRNA feeding of other organisms can use our parameters as a starting point for their efforts.
Table 1. Comparison of RNAi Methodologies Used in Planarian Researcha
|RNAi methodology||Robustness||Ease of use||Quality control|
|In vitro feeding||+||+||+|