What is RNA Interference (RNAi)?

• RNA interference (RNAi) is a technique that “turns off” or “knocks down” specific genes in organisms.
• In this experiment, RNAi is used to study how specific genes in planarians (a type of flatworm) function during regeneration (regrowing lost parts) and tissue maintenance.
• RNAi works by injecting double-stranded RNA (dsRNA) into the organism, which triggers the cells to break down messenger RNA (mRNA) for the targeted gene, preventing it from being expressed.

Why Planarians?

• Planarians are a great model for studying regeneration because they can regrow lost body parts, like tails or heads.
• This ability is controlled by genes, and RNAi helps scientists study how different genes affect regeneration and healing.

Overview of the RNAi Procedure

• The process starts by creating the dsRNA needed to target the gene you want to “turn off.”
• The dsRNA is then injected into the planarians, where it will interfere with the gene’s expression.
• After injection, the effects of the RNAi are observed, looking for changes in the planarian’s ability to regenerate or maintain tissues.

Step-by-Step Method

• Step 1: Prepare RNA for Injection
  • Start by assembling two transcription reactions (one with T3 polymerase and one with T7 polymerase).
  • Each reaction contains DNA, ribonucleotides, and other chemicals necessary for creating RNA.
  • Incubate these reactions at 37°C for 2 hours.
  • After incubation, treat the RNA samples with DNase to remove any leftover DNA.
  • Purify the RNA using a series of centrifugation and wash steps to remove impurities.
  • Resuspend the final RNA in water to prepare for injection.
• Step 2: Prepare the Microinjection Needles
  • Use a micropipette puller to form the needles.
  • Break the tip of the needle carefully under a dissecting microscope to create a small opening, ensuring the liquid can exit easily but not too quickly.
  • Fill the needle with mineral oil to avoid air bubbles.
• Step 3: Inject the dsRNA into the Planarian
  • Place the planarian on cold, wet tissues to keep it moist during the procedure.
  • Using the microinjector, carefully insert the needle into the planarian and inject the dsRNA.
  • Multiple injections are needed (usually 3-5) to ensure the dsRNA reaches all parts of the planarian.
  • The injected dsRNA should fill the planarian’s gastrovascular system, confirming that the injection was successful.
• Step 4: Monitor the Effects
  • After the injections, observe the planarians for any changes, especially in their ability to regenerate or heal.
  • Sometimes, it may take multiple injections over days or weeks to see strong effects.

Troubleshooting Tips

• Problem 1: RNA is not visible on the gel.
  • Make sure that all reagents are fresh and of high quality.
  • Check that the DNA template has the correct T3 and T7 polymerase promoters.
  • Ensure all equipment is clean and free of contaminants.
• Problem 2: No liquid comes out of the needle during injection.
  • Ensure there are no air bubbles in the needle.
  • Confirm the needle is correctly attached to the microinjector.
• Problem 3: You’re not sure if the liquid is inside the planarian.
  • Add a few microliters of food coloring to the injection solution to help track whether it’s being injected correctly.
• Problem 4: No observable phenotype (change) in the planarians.
  • Check if the target gene is being effectively “knocked down” using methods like in situ hybridization or RT-PCR.
  • Consider adjusting the injection schedule or targeting multiple genes if necessary.

Key Conclusions

• RNAi is a powerful tool for studying gene function in planarians, particularly in the context of regeneration and tissue maintenance.
• While the technique requires careful preparation and injection, it can yield valuable insights into how specific genes control the ability of planarians to regenerate.
• It’s important to monitor the planarians over time to fully assess the impact of the RNAi treatment.

什么是 RNA 干扰 (RNAi)？

• RNA 干扰 (RNAi) 是一种技术，通过它可以“关闭”或“降低”特定基因的表达。
• 在这个实验中，RNAi 被用来研究特定基因在计划虫（一种扁形虫）再生（重新长出丢失的部分）和组织维持中的作用。
• RNAi 通过将双链 RNA (dsRNA) 注入生物体内，触发细胞分解靶基因的信使 RNA (mRNA)，从而阻止其表达。

为什么选择计划虫？

• 计划虫是一种很好的再生研究模型，因为它们能够重新长出失去的身体部分，如尾巴或头部。
• 这种能力是由基因控制的，RNAi 可以帮助科学家研究不同基因如何影响再生和愈合。

RNAi 程序概述

• 该过程从创建需要的 dsRNA 开始，靶向你想要“关闭”的基因。
• 然后将 dsRNA 注入计划虫中，它将干扰基因的表达。
• 注射后，观察 RNAi 的效果，查看计划虫在再生或维持组织方面的变化。

详细步骤

• 步骤 1: 准备 RNA 进行注射
  • 开始时，组装两个转录反应（一个使用 T3 聚合酶，一个使用 T7 聚合酶）。
  • 每个反应中包含 DNA、核糖核苷酸和其他合成 RNA 所需的化学物质。
  • 将这些反应在 37°C 下孵育 2 小时。
  • 孵育后，用 DNase 处理 RNA 样本，以去除任何残留的 DNA。
  • 通过一系列离心和清洗步骤纯化 RNA，以去除杂质。
  • 将最终的 RNA 重悬于水中，为注射做准备。
• 步骤 2: 准备微注射针
  • 使用微量注射器拉制针头。
  • 在解剖显微镜下小心地断开针头，制造一个小孔，确保液体能够顺利流出，但不要太宽。
  • 用矿物油填充针头，避免气泡。
• 步骤 3: 将 dsRNA 注入计划虫
  • 将计划虫放在冷湿的纸巾上，保持它在整个过程中湿润。
  • 使用微注射器，小心地将针头插入计划虫体内，并注射 dsRNA。
  • 需要多次注射（通常是 3-5 次）以确保 dsRNA 到达计划虫的各个部分。
  • 注射的 dsRNA 应该填满计划虫的胃肠系统，确认注射成功。
• 步骤 4: 观察效果
  • 注射后，观察计划虫是否有变化，特别是在再生或愈合方面。
  • 有时可能需要多次注射，或者注射数天或数周后才能看到强烈的效果。

故障排除提示

• 问题 1: 在凝胶上看不到 RNA。
  • 确保所有试剂都是新鲜的并且质量良好。
  • 检查 DNA 模板是否包含正确的 T3 和 T7 聚合酶启动子。
  • 确保所有设备清洁且无污染物。
• 问题 2: 注射针中没有液体流出。
  • 确保针头内没有气泡。
  • 确认针头是否正确连接到微注射器。
• 问题 3: 不确定液体是否进入计划虫。
  • 可以向注射溶液中加入几微升食用色素，以帮助追踪注射是否正确。
• 问题 4: 计划虫没有表现出任何表型（变化）。
  • 检查靶基因是否通过原位杂交或 RT-PCR 有效“降低”。
  • 考虑调整注射时间表或靶向多个可能相互补偿的基因。

关键结论

• RNAi 是研究计划虫基因功能的强大工具，特别是在再生和组织维持方面。
• 虽然该技术需要小心的准备和注射，但它可以为科学家提供有关基因如何控制计划虫再生的宝贵信息。
• 观察计划虫一段时间，才能完全评估 RNAi 处理的影响。