Introduction (What is this Protocol About?)

• This protocol explains how to reduce or “knockdown” a gene’s activity in planarians using RNA interference (RNAi).
• Planarians are flatworms commonly used to study regeneration (the process of regrowing lost body parts) and tissue maintenance.
• By injecting lab-made double-stranded RNA (dsRNA) into the planarian, scientists can lower the expression of a specific gene and observe resulting changes, called phenotypes.
• This method helps researchers understand the role of different genes during regeneration.

Related Background Information

• The technique was originally described by Sánchez Alvarado and Newmark in 1999.
• Planarians serve as a powerful model system for studies on regeneration, adult stem cell regulation, aging, and behavior.
• Other protocols related to planarians include methods for maintaining colonies and imaging their membrane potential.

Key Terms and Concepts

• RNA interference (RNAi): A method used to reduce or silence the expression of a specific gene.
• Double-stranded RNA (dsRNA): Two complementary strands of RNA that, when introduced into an organism, trigger RNAi.
• Phenotype: The observable traits or changes in an organism that result from a change in gene activity.
• Microinjection: A technique for delivering small volumes of liquid into an organism using a very fine needle.
• In vitro: Procedures performed in a controlled laboratory environment outside a living organism.

Step-by-Step Method: RNA Synthesis

• Prepare two separate transcription reactions using enzymes:
  • One reaction uses T3 polymerase and the other uses T7 polymerase.
  • Each reaction includes: DNA template (1 μg), DTT (10 mM), ribonucleotides (1%), RNA transcription buffer (20%), RNasin (60 units), and the respective polymerase (17 units).
  • If using a vector with two T7 promoters, a single T7 reaction may be used after linearizing the DNA.
• Incubate both reactions at 37°C for 2 hours to synthesize RNA.
• Add DNase I (1 unit) to each reaction and incubate at 37°C for 15 minutes to remove the DNA template.
• Reserve 1 μL from each reaction in separate tubes and store at -20°C for later comparison.
• Combine the remaining 19 μL from each reaction into one tube.
• Add 360 μL of RNAi Solution A to the combined reaction and let it sit at room temperature for 10 minutes.
• Add 200 μL of a phenol:chloroform mixture and vortex vigorously to mix.
• Centrifuge at 14,000 rpm for 2 minutes at room temperature and transfer the clear aqueous phase to a new tube.
• Add 200 μL of chloroform, vortex again, and centrifuge at 14,000 rpm for 2 minutes; transfer the new aqueous phase to another fresh tube.
• Heat the tube in a water bath at 68°C for 10 minutes to denature (unfold) the RNA, then incubate at 37°C for 30 minutes to allow the RNA strands to reanneal and form dsRNA.
• Add 1 mL of cold 100% ethanol and centrifuge at 14,000 rpm for 15 minutes at 4°C to precipitate the RNA.
• Discard the supernatant and wash the RNA pellet with 1 mL of cold 80% ethanol; centrifuge at 14,000 rpm for 10 minutes at 4°C.
• Discard the wash solution and resuspend the RNA pellet in 10 μL of nuclease-free water; keep the sample on ice.
• Verify RNA quality and confirm dsRNA formation by running a small amount on a 1% agarose gel under non-denaturing conditions.

Step-by-Step Method: Microinjection of dsRNA

• Prepare the microinjection needle:
  • Use a micropipette puller to form a long, thin needle.
  • Break the tip slightly under a dissecting microscope so that about 10%-25% of the tip is removed; the opening should be small enough to prevent unwanted leakage.
• Fill the needle with mineral oil, ensuring that no air bubbles are present.
• Attach the needle to the microinjector and adjust its position under a dissecting microscope:
  • Set the microinjector to deliver 32 nanoliters (nL) per pulse.
• Aspirate 1–2 μL of the dsRNA solution into the needle.
• Place the planarian on cold, wet tissue (placing ice underneath helps keep it cool and still).
• Carefully insert the needle into the planarian, typically near the prepharyngeal area (just in front of the mouth), to ensure the needle tip enters the body.
• Press the injection key to dispense 32 nL; repeat this 3 to 5 times so that the worm’s gastrovascular system fills with the dsRNA solution, confirming successful injection.
• Transfer the injected planarian to a Petri dish containing fresh planarian water at room temperature.
• For a stronger gene knockdown effect, repeat the injection process over consecutive days or weeks.

Troubleshooting Common Problems

• If RNA (ssRNA or dsRNA) is not visible on the agarose gel:
  • Check that all reagents are fresh and have not been repeatedly frozen and thawed.
  • Ensure the DNA template includes both T3 and T7 promoter regions.
  • Work on clean surfaces free from RNase and DNase contamination.
• If no liquid is dispensed from the needle during injection:
  • Examine the needle for air bubbles.
  • Ensure the needle is securely attached to the microinjector.
• If you are uncertain whether the injected liquid is entering the planarian:
  • Practice the injection technique; adding a small amount of food coloring to the injection solution can help visualize the process.
• If no observable phenotype is produced:
  • Check the gene knockdown by methods such as in situ hybridization or RT-PCR to confirm reduced gene expression.
  • Consider adjusting the injection schedule or targeting two genes that may compensate for each other.

Materials and Equipment

• Reagents:
  • 1% agarose gel, DNA template (with T3 and T7 promoters), DNase I, DTT, ribonucleotides, RNA transcription buffer, RNasin, T3 and T7 polymerases, nuclease-free water, phenol:chloroform mix, chloroform, 100% and 80% ethanol, and RNAi Solution A.
• Equipment:
  • Microcentrifuge (with both room temperature and 4°C capability), water baths, vortex mixer, micropipette puller, microinjector, dissecting microscope, Petri dishes, and apparatus for agarose gel electrophoresis.
• Planarians (the flatworms) and planarian water for maintaining them.

Acknowledgments and References

• The authors acknowledge contributions and funding from organizations such as the NIH, NSF, and others.
• For more detailed information, refer to the publications by Oviedo et al. (2008) and Sánchez Alvarado and Newmark (1999).

Chinese Version: 简介 (研究背景)

• 本协议描述了如何利用RNA干扰（RNAi）在平片虫中实现基因敲低，即降低某个基因的活性。
• 平片虫是一种常用于研究再生（失去的部位重新生长）和组织维持的扁形动物。
• 通过向平片虫注射体外合成的双链RNA（dsRNA），科学家可以降低目标基因的表达，从而观察到由此产生的表型变化。
• 这种方法有助于研究者理解不同基因在再生过程中的作用。

相关背景信息

• 该技术最早由 Sánchez Alvarado 和 Newmark (1999) 描述。
• 平片虫被认为是研究再生、干细胞调控、衰老及行为的重要模型系统。
• 其他相关协议包括建立平片虫种群及平片虫细胞膜电位的成像方法。

关键术语和概念

• RNA干扰 (RNAi)：一种降低或沉默特定基因表达的方法。
• 双链RNA (dsRNA)：由两条互补RNA链组成的分子，用于触发RNAi反应。
• 表型：基因活性改变后在生物体上表现出的可观察特征。
• 微注射：使用极细针头向生物体内注入极少量液体的技术。
• 体外：在实验室内控制环境中进行的实验操作，而非在生物体内进行。

分步方法：RNA合成

• 准备两个独立的转录反应体系，分别使用T3和T7聚合酶：
  • 每个反应中加入：DNA模板 (1 μg)、DTT (10 mM)、核糖核苷酸 (1%)、RNA转录缓冲液 (20%)、RNasin (60单位)和相应的聚合酶 (17单位)。
  • 如果使用双T7启动子的载体，可在酶切后只用T7聚合酶分别进行反应。
• 在37°C水浴中孵育2小时以进行RNA合成。
• 向每个反应中加入1单位DNase I，并在37°C下孵育15分钟以去除DNA模板。
• 从每个反应中取出1μL样品，另存备用并置于-20°C。
• 将剩余的反应混合物合并到一个管中。
• 加入360μL RNAi专用溶液A，在室温下静置10分钟。
• 加入200μL酚:氯仿混合物，剧烈震荡混合以提取RNA。
• 在室温下以14000 rpm离心2分钟，将上层水相转移到新管中。
• 加入200μL氯仿，震荡混合后以14000 rpm离心2分钟，再次转移上层水相到新管中。
• 在68°C水浴中加热10分钟使RNA变性，再于37°C水浴中孵育30分钟使RNA重新配对形成双链RNA。
• 加入1 mL冷却的100%乙醇，在4°C下以14000 rpm离心15分钟以沉淀RNA。
• 弃去上清液，用1 mL冷80%乙醇洗涤RNA沉淀，再以14000 rpm在4°C离心10分钟。
• 弃去洗涤液，将RNA沉淀重悬于10μL无RNA酶水中，并置于冰上保存。
• 通过在1%琼脂糖凝胶上检测1μL ssRNA样品和0.5μL dsRNA样品，确认双链RNA的形成。

分步方法：dsRNA的微注射

• 准备微注射针：
  • 使用微吸管拉针仪制作细长针头。
  • 在解剖显微镜下观察，轻轻敲碎针尖，去除大约10%-25%的针尖，确保开口足够小以防止液体泄漏。
• 用矿物油填充针头，确保无气泡。
• 将针头安装到微注射器上，并在解剖显微镜下调整好位置：
  • 设定每次注射量为32纳升 (nL)。
• 吸取1-2μL dsRNA溶液（来自RNA合成步骤）。
• 将平片虫放在冷湿纸巾上（在下面放冰可保持其凉爽和湿润）。
• 小心将针头插入平片虫，通常选择口前区域，确保针头进入体内。
• 按下注射键释放32纳升液体，重复3至5次，使平片虫的胃血管系统充满dsRNA溶液，以确认注射成功。
• 将注射后的平片虫转移到含有新鲜平片虫水的培养皿中，并保持室温。
• 为增强表型效果，可在连续几天或几周内重复注射。

常见问题及解决方案

• 若在琼脂糖凝胶上未检测到RNA（ssRNA或dsRNA）：
  • 检查所有试剂是否新鲜，且未反复冻融。
  • 确认DNA模板包含T3和T7启动子区域。
  • 确保在无RNase和DNase污染的环境下操作。
• 若注射时针头无液体流出：
  • 检查针头内是否存在气泡。
  • 确认针头是否正确安装在微注射器上。
• 若不确定液体是否进入平片虫体内：
  • 多加练习，可在注射液中加入少量食用色素以便观察。
• 若未观察到任何表型变化：
  • 通过原位杂交或RT-PCR检测目标基因表达是否降低。
  • 考虑调整注射时间表，或同时靶向两个可能互补的基因。

材料与设备

• 试剂：
  • 1%琼脂糖凝胶、含T3和T7启动子的DNA、DNase I、DTT、核糖核苷酸、RNA转录缓冲液、RNasin、T3和T7聚合酶、无RNA酶水、酚:氯仿混合物、氯仿、100%及80%乙醇、RNAi专用溶液A。
• 设备：
  • 微量离心机（水浴室温和4°C均可）、水浴、震荡器、微吸管拉针仪、微注射器、解剖显微镜、培养皿及琼脂糖凝胶电泳仪器。
• 平片虫及用于维持平片虫生长的平片虫水。

致谢与参考文献

• 作者感谢各资助机构（如NIH、NSF等）及相关人员的支持。
• 更多详情请参阅 Oviedo 等人 (2008) 以及 Sánchez Alvarado 与 Newmark (1999) 的相关文献。