Gene expression plays a pivotal role in the development, differentiation, and maintenance of organisms by allowing genes to encode for an observable trait (i.e., phenotype). To understand the function of a particular gene, several approaches can be taken, ranging from removing the gene entirely to targeting the product of the gene (i.e., the protein). RNA interference (RNAi) has been shown to be a powerful approach used to silence gene activity and examine the connection between DNA and protein along with controlling gene expression. The course-based undergraduate research experience (CURE) described in this article is a hands-on molecular biology lab–based lesson that allows students to examine the impact of RNAi on Caenorhabditis elegans reproduction and development through the examination of the central dogma of biology. Through these activities, students gain practice in the scientific method of inquiry by designing experiments to observe how genotype connects to phenotype and, subsequently, organism behavior.
The primary goal of any course-based undergraduate research experience (CURE) in the natural sciences is to provide an opportunity to integrate teaching and research in a traditional undergraduate lab to foster scientific inquiry and collaboration (Burmeister et al., 2021). Incorporating independent research projects into a course curriculum requires students to take the knowledge learned from lower-division courses and move up the ladder of higher-order thinking to apply it to a scientific conundrum they find interesting. Studies have shown that hands-on inquiry-based activities not only help students further develop their critical thinking and problem-solving skills, but also have a positive impact on student learning and build confidence in carrying out basic laboratory techniques (Pavlova et al., 2021). Students develop a sense of ownership around their proposed projects, which authenticates the independent research experience.
The CURE described in this article is meant to provide a framework for students to explore the scientific method of inquiry using the nematode Caenorhabditis elegans and become proficient in fundamental molecular biology. Caenorhabditis elegans are soil-dwelling microscopic nematodes that feed on bacteria in the wild. Due to their transparency, size (~1 mm in length), short larval stages, and small genome size as well as the ease of culturing them, they serve as an excellent model organism with which to examine the role of genes of interest and their homologs in humans (Meneely et al., 2019). In addition, because C. elegans are invertebrates, research ethics approval is not required, making them ideal for an undergraduate lab curriculum and less likely to generate emotional reactions from students. For decades, C. elegans have been used to study neuronal growth and development, ion channel sensitivity and function, memory and plasticity, DNA structure and repair, cell contact and organ formation, and more (Apfeld & Alper, 2018).
Students come away from the project with the understanding that many questions remain unanswered … and that the experimental process requires troubleshooting, time, and perseverance.
One approach to examining gene function in C. elegans growth and development is the introduction of RNA-mediated interference (RNAi) (Fire et al., 1998; Maine, 2008). RNAi is a well-established biological process that works to silence gene activity through transcriptional and/or translational repression. For additional background information on the RNAi mechanism of action and its impact on biological function, see Grishok (2005) and Agrawal et al. (2003). Applications of RNAi range far and wide, from the treatment of various human diseases to genetic engineering of food and crops for human consumption (Ibrahim & Aragão, 2015; Wittenburg et al., 2000). Introduction of an inducible DNA plasmid expressing double-stranded RNA (dsRNA) for a gene of interest inactivates gene activity by targeting the endogenous messenger RNA (mRNA) (Conte et al., 2015). Several effective methods have been used to introduce dsRNA (i.e., RNAi induction) into C. elegans, and because of their ability to produce results within 72 hours after RNAi induction, they make a prime model organism to use in a classroom lab setting (Andersen et al., 2008). By introducing dsRNA plasmids containing genes of interest into C. elegans, one can examine the connection between phenotype (observable trait) and genotype (genetic makeup), explore the central dogma of biology to understand gene expression (DNA → mRNA → protein), and begin to appreciate the elaborate connection (and redundancy) of genes to organism development and function.
The lab activities laid out in the current article are designed to be an iterative process, where students practice established techniques on a given model system, perform experiments, tweak, and refine their lab procedures to generate data, and report out their results in an oral and written fashion (Light et al., 2019). This approach allows the instructor to provide a transformative lab experience and for students to have an authentic research experience within a structured classroom setting.
Lab Learning Outcomes
After completing the following activities, students will be able to:
Examine the RNAi mechanism in C. elegans to understand the connection between genotype and phenotype.
Develop a research question and hypothesis related to a gene of interest and design experiments to test the hypothesis.
Identify and evaluate relevant primary literature and background information related to the project.
Understand how to use logic and evidence to build arguments and draw conclusions from collected data.
Demonstrate how to effectively communicate research findings in oral and written scientific formats.
Materials and Methods Overview
The lab activities for this CURE take place over a 14-week semester and are divided into two parts with students working in small groups. Part I involves students examining the RNAi molecular mechanism in C. elegans to make the connection between phenotype and genotype, using established protocols from a Carolina Biological kit. This allows both instructor and students to explore C. elegans biology and development together with a known outcome. Part II involves students taking the RNAi techniques from Part I and applying those techniques to a chosen gene of interest (GOI) selected by the student group. Each group replicates what has already been published on the chosen GOI and/or discovers a different phenotype when RNA levels have been reduced. For an overview of the experimental process in C. elegans, see Figure 1. For additional information on materials, recipes, cost, protocols, and genes of interest to consider for the following procedures, see Appendix 1 in the Supplemental Material available with the online version of this article.
Part I: Introduction to the RNAi Mechanism
For students to become familiar with not only the care and maintenance of C. elegans, but also the major steps involved in silencing genes in C. elegans, all groups spend the first six weeks of the semester following the step-by-step protocol in the Examining the RNAi Mechanism kit from Carolina Biological Supply (Figure 2). The kit explores how the introduction of double-stranded RNA (dsRNA) in C. elegans can lead to inactivation of a particular gene through the degradation of endogenous mRNA. Students are introduced to the RNA mechanism by observing wild-type (N2) and mutant (e.g., dumpy 13, or dpy-13) worms to become familiar with worm development, growth, movement, and appearance. In addition, students are exposed to the basics of bioinformatics and learn how to navigate the National Center for Biotechnology Information (NCBI) and worm-based database (i.e., WormBook) sites to examine the dpy-13 gene and resulting protein in the worm. Students start the experiment knowing the phenotype they are looking for in RNAi-treated worms and learn to describe the difference between negative, positive, and experimental treatment groups for comparison.
One advantage of the kit is that the reagents are designed for the project to work if students follow the instructions carefully. Included in the kit are worksheets for students to complete, to facilitate their understanding of the designed experiment. Students learn the basics of DNA isolation, polymerase chain reaction (PCR), and gel electrophoresis, and they observe firsthand the amount of time required to follow through with the experiment and achieve results. The worksheet and questions associated with each step of the protocol enhance the students’ appreciation for the targeting effect of RNAi on the worm, especially when they can replicate the “dumpy” phenotype in the RNAi treated group. One disadvantage is that students usually have a difficult time grasping the molecular impact of RNAi on RNA levels, because the kit only has students examine DNA levels to draw conclusions about whether the RNAi treatment was effective in isolated worms.
To help students understand the impact of the dpy-13 dsRNA feeding strain on mRNA levels, the Carolina Biological kit is modified by adding a couple more steps to the protocol. This includes selecting worms from the various treatment groups to isolate RNA using the Monarch Total RNA Miniprep Kit. Students work through the process of converting RNA to complementary DNA (cDNA, a double-stranded version of mRNA and a measure of how much mRNA is present) using the First Strand cDNA Synthesis Kit and reverse transcription–polymerase chain reaction (RT-PCR) protocol. Subsequently, agarose gels are loaded with similar amounts of cDNA to determine if the RNAi treatment had a significant impact on the dpy-13 RNA product. Using this approach, students work through the differences between DNA, mRNA, and cDNA and how each contributes to the process of determining whether their particular gene product (i.e., dpy-13) was reduced in RNAi-treated worms (e.g., connecting genotype to phenotype). Furthermore, students learn a series of techniques that they can now apply to an independent research project on a different gene in the worm.
Part II: Examining the RNAi Mechanism through an Independent Group Research Project
The group-driven independent research in the second half of the semester project provides students with the chance to apply their newly acquired molecular biology techniques to a different gene in the worm. The Horizon company has a database of dsRNA bacterial feeding strains for many C. elegans genes cloned into a pL4440-DEST vector. The instructor picks 10 genes from the collection that may or may not have a strong observable phenotype. In a classroom setting, this approach allows students to make the connection between phenotype and genotype and/or learn how genetic redundancy can cause a no-phenotype knockdown effect. Each group works to select a gene from the list of 10 for their independent research project (see Appendix 1 in the Supplementary Material available online for a list of genes with observable phenotypes). The remaining weeks of the semester are dedicated to the selected gene of interest (GOI). Components of the flowchart in Figure 2 are outlined to provide additional context for the group independent research project.
Week 6. Students work in their established groups to do background literature searches on their GOI. Based on the knowledge they gather, they work to design a research question and hypothesis centered around their background research on the GOI. Students are required to complete a Research Question and Hypothesis worksheet (see Appendix 2 in the Supplementary Material available online). The worksheet provides a way for the instructor to (1) give initial feedback to each group, (2) help students understand the difference between question and hypothesis, and (3) ensure students have a solid question and hypothesis before moving forward with the proposal portion of the project.
Week 7. Once each group has identified a GOI, one class session is dedicated to learning how to design primers to identify their gene product at the molecular level. The primer design activity provides students with the “know-how” on how to properly design primers (see Appendix 1 in the Supplemental Material online for a Primer Design Tips link) to achieve optimal results. Students use a primer design program through the company Integrated DNA Technologies (IDT) to design the best primer pair for their GOI (see Appendix 1 for a PrimerQuest Tool link). Instructors are encouraged to have students test out the generated primers to ensure that the designed primers yield the predicted GOI PCR amplicon length/size.
Week 8. Each group designs and submits a two-to-three-page initial research proposal, using a rubric and set of guidelines (see Appendix 3 in the Supplementary Material online for details). Typically, students spend weeks 6–8 on the proposal. The instructor is encouraged to give feedback on the proposal design to ensure that students (1) select the proper experimental techniques to collect data that will inform their project hypothesis and (2) consider all the potential results that either support or do not support the project hypothesis.
Weeks 9–13. Groups work on the proposed project experiments, using techniques similar to those in Part I, but now examining a different GOI. For the independent research portion, each group is provided with a different wild-type C. elegans worm strain, rrf-3. This “wild-type” strain has been genetically engineered to be highly sensitive to RNAi treatment (Simmer et al., 2002). In Part II, students use OP50 bacteria as their negative RNAi control, as in Part I. However, an empty pL4440-DEST vector in a bacterial feeding strain would serve as a proper negative RNAi control to demonstrate that the RNAi silencing mechanism is due to the presence of the gene. Each group performs various molecular biology techniques, which include bioinformatic searches, DNA and RNA isolations, PCR and RT-PCR, and gel electrophoresis. Students are required to collect two pieces of evidence from their group project (see an example of data generated by students in Figure 3). One example of required phenotypic evidence students provide is an image of the preliminary phenotype in the C. elegans and/or quantification of a possible outcome from knocking down the GOI (e.g., body bends and/or spontaneous reversals to examine locomotion defects) to determine if the RNAi effect causes a disruption to C. elegans development, growth, and/or behavior. These types of functional assays are impactful if the GOI being examined is connected to muscle/nerve development such as the WASP (actin cytoskeleton modulator) homologous gene, wsp-1, involved in regulating the actin cytoskeleton in C. elegans (Figure 3A–B). For the experiment described in Figure 3, six individual worms from the control and RNAi-treated plates were measured for the body bends and reversals data from one RNAi induction. Additional worms should be scored to validate the behavior phenotype (minimum 20–30 worms per treatment). For more information on how to set up and control for behavior-based assays, see the Behavior section of the WormBook site (Hart, 2006).
The genotypic piece of evidence required is an image of an agarose gel displaying DNA and cDNA expression products of the GOI to determine if the RNAi was successful. The DNA agarose gel is a critical component of the group project, as it shows whether the dsRNA for the GOI was effective in reducing the amount of mRNA (i.e., cDNA) of the targeted gene. Preliminary results in Figure 3C suggest that the RNAi (dsRNA) feeding strain for wsp-1 successfully reduced the amount of wsp-1 cDNA (mRNA) present in worms—compare RNAi-treated versus WT control (OP50) lanes. DNA levels were unaffected, which shows that RNAi treatment effectively targets mRNA, not DNA. To confirm that the RNAi feeding strain targeted wsp-1, students also examined a housekeeping gene that should not be targeted by the RNAi treatment. As indicated in Figure 3C, ama-1 (RNA polymerase gene) expression levels were unaffected in both WT control (OP50) and RNAi-treated worms. Students may have to troubleshoot the PCR conditions for their GOI to achieve a final product; most groups achieve the correct amplicon length/size for their GOI on isolated DNA. It has proved to be somewhat difficult at times to get a detectable amount of cDNA PCR product at the correct length/size for RT-PCR experiments; however, most groups have achieved varied success (as demonstrated in Figure 3C—compare cDNA from WT (OP50) lanes and RNAi-treated lanes). The instructor should continually remind students that RNA is not stable and that proper collection and handling procedures must be followed to achieve a final product.
Week 14. In the final week of lab, students are required to present their findings in an oral presentation to the instructor and their peers, using PowerPoint or Google Slides (see Appendix 4 in the Supplementary Material online for presentation guidelines and a rubric). In addition to the presentation, each group modifies their research proposal to include their preliminary results/findings from their proposed experiments and discusses these findings in the context of their research question(s) and hypothesis along with future directions. The written products described above are submitted on Turnitin.com to monitor for originality.
The inquiry-based lab activities described in this article are meant to guide instructors on how to implement an RNAi-based technique in an upper-division molecular biology, genetics, or cell biology course where students have learned the fundamentals of gene expression in a lower-division biology course for majors. Parts I and II described above are typically carried out in an upper-division undergraduate molecular biology lab course of 14 to 16 students. Students have the opportunity to not only analyze and evaluate data, but possibly generate new data on a particular gene and find ways to explain their results in the context of what is currently in the literature. Furthermore, the inclusive curriculum design provides students with some form of research experience, especially students who are unable to complete independent research projects with faculty outside of a traditional classroom lab setting (Bangera & Brownell, 2014).
From the activities described, several goals are achieved. First, students have a much better appreciation for the flow of information (DNA → RNA → protein) and the impact of RNAi on gene expression. Students appreciate firsthand how genotype is connected to phenotype and that disruption of only one gene can have a major impact on worm development and function. The iterative process students go through as they learn basic molecular biology techniques (e.g., DNA/RNA isolations, PCR) provides a critical step in their development as scientists, to refine their ideas and practice applying new skills to different questions surrounding worm development (Corwin et al., 2018).
A second goal of the inquiry-based lab activities is to help students develop oral and scientific writing skills (Brownell et al., 2013). A critical part of the project in Part II requires students to come up with a question and hypothesis based on background data on a particular gene. By developing a solid hypothesis, students learn how to anticipate possible outcomes from the experiment. It is imperative for instructors to remind students of the importance of revisiting their question and hypothesis throughout the process, to ensure their data are in alignment with what they set out to accomplish. By doing this, students further hone their critical thinking skills and learn how to connect their hypothesis to the overarching importance of the gene to C. elegans development and function. The proposal, research paper, and oral presentation challenge students to describe their justifications for why they have chosen a particular GOI and practice fielding questions based on their project design and discovery. These activities build self-confidence and provide the necessary scientific communication skills to help prepare students to take on other science courses in their major or higher degree programs.
A final goal of the inquiry-based lab activities is to further ingrain within students the importance of project design and time management skills. Students learn the time frame for the lab activities in Part I and translate this timeline to an independent research project where they build the design and structure of the project based on their hypothesis. In addition, by working in groups, students build leadership and effective communication skills within their peer groups to not only carry out the experiments on a weekly basis, but also be effective listeners and team players in the process (Esparza et al., 2020). Many of these skills will eventually translate to their future careers once they graduate and move on to the next phase of their educational journeys.
Overall, this lab design provides a hands-on approach to understanding how gene structure and expression connects to protein function and organism development. At the end of the semester, students come away from the project with the understanding that many questions remain unanswered when it comes to the role of genes in growth and development and that the experimental process requires troubleshooting, time, and perseverance.
Appendix 1. RNAi Materials and Protocols List
Appendix 2. Background Information and Hypothesis/Research Question
Appendix 3. Research Proposal Guidelines and Rubric
Appendix 4. Research Proposal Presentation Guidelines