这项研究已被授予NIHM MH091816 R01和R21的MH084043到TDG支持。

1。材料 1。暂停盒尾部悬吊试验（TST）涉及暂停小鼠，它们的尾巴地面以上。在最基本的层面上，这个过程只需要吊杆货架窗台，和磁带。然而，实验者应考虑使用一个背景下，提供最佳的对比度。此外，它是审慎采取措施，以防止小鼠观察正在测试的其他动物。在我们的实验室，我们使用专门制造的尾吊箱（四小时日，巴尔的摩MD），由塑料制成的尺寸（60 55高X宽X深11.5厘米）。为了防止动物观察或相互作用彼此，每个鼠标悬浮在自己的薄壁矩形盒（55高× 15宽x 11.5厘米深度）。鼠标悬浮在车厢中的宽度和深度是足够的大小，使鼠标不能使合作ntact与墙壁。在此设置中，老鼠的鼻子和仪器地板之间的大致距离为20-25厘米。有四个相同的仪器车厢，让我们一次测试四只老鼠。暂停每个鼠标的尾部，铝吊杆（1厘米。高度x 1厘米× 60厘米，宽度长度），定位在框的顶部。在我们的实验室中使用的尺寸，我们应该算是作为一般参考。例如，个别车厢的尾部悬浮框的大小可以增加，如果一个大的远交小鼠品系（如CD - 1）是使用。 我们在每个车厢的底部放置一个可拆卸的铝制托盘收集动物粪便或尿液。我们用一个白化动物的暗灰色方块和其他毛色的小鼠奶油色的框。这样的安排，为我们提供了更好的对比度和行为因此更可靠的测试得分。 2。磁带 3。定时器 4。录像设备一台摄像机和三脚架（或其他支持结构）是必要的。由于这种测试通常涉及多个动物在同一时间测试，现场打分将是困难的，不建议。摄像头应该记录在嗨GH足够的分辨率来呈现高质量的画面，行为得分后，将用于。务必一定是有足够的录音内存在开始测试前的相机。我们使用一台摄像机，记录数字没有使用的物理介质（如录像带），允许简单的视频传输。 5。白噪声发生器噪声发生器应屏蔽间歇性的环境声音。特别推荐突然大声喧哗，可以听到，可能不惊人死不休小鼠在实验室环境噪声发生器的使用。在我们的试验室的环境噪声水平（不带白噪声发生器激活）约60分贝。在尖沙咀进行的位置激活与白噪声发生器总声级为70-72分贝。但是，应该指出的是，这些数字作为例子只提供，每个实验室应选择根据其独特的环境和情况下，正确的噪音水平。 6。清洁用品每届会议结束后应清除悬浮框，彻底灭菌解决方案（例如，MB - 10，妙语Laboratories公司，威尔明顿，DE或类似）。 7。 Climbstoppers（可选:使用后应变依赖） 一些背景，如C57BL / 6小鼠的尾巴爬在测试1。清除空心圆柱体（4厘米长，1.6厘米外径，内径1.3厘米，1.5克），切成4厘米长，由聚碳酸酯管（FourHourDay公司，巴尔的摩MD）（＃8585K41，麦克马斯特卡尔，圣塔菲泉，CA）的周围放置老鼠的尾巴，以防止这种尾登山行为2,3。这些设备可以在任何实验室，与常用的工具和材料。 2。行为程序<ol><li>将相机位置。相机应尽可能靠近，以获得最高的动物可能的解决方案。 </li><li>将在会议期间使用的磁带碎片切割，标记，并为本届会议准备。 </li><li>开始白噪声发生器，如果正在使用，小鼠之前推出的试验室。白噪声水平只应足以屏蔽外部噪声。避免以高音量，并确保所有动物中使用的同一级别的白噪声。 </li><li>带来的一个imals进入测试室。如果房间里的动物所在考场毗邻与其他类似的环境条件，没有驯化期间可能是必要的。否则，放置在试验室动物的驯化期（一般至少一小时）。请注意，放置在同一个房间里的其他动物可以感受到嗅觉和超声波线索。 </li><li>如果攀登它们的尾巴，如C57BL6 / J，应变，尾巴周围Climbstoppers之前申请的磁带。 </li><li>老鼠的尾巴坚持磁带。使用标记的磁带结束，并确保磁带坚持回到自己的尾巴和。应适用于磁带的尾巴很结束与2-3毫米磁带以外的剩余尾。 </li><li>一旦每一块的磁带连接到鼠尾笼内壁粘磁带的中间部分。这将有助于防止相互缠绕磁带件。请注意，confinement以这种方式可能造成的压力和重要的是要尽快磁带每个动物的过程中完成。 </li><li>一旦适用于所有的磁带，开始录制前小鼠暂停，并确定了会议。 </li><li>暂停吊杆或架子上，在治疗组之间平衡的顺序放置磁带的动物。暂停在小鼠的方式，是因为整个尖沙咀会议将取得和阻挠相机会导致无法评估在这段时间内的行为不妨碍相机视图。检查所有磁带两端都挂平行的懈怠每个鼠标类似长度的吊杆或架子。 </li><li>在会话结束（通常是6分钟），返回的动物，他们homecage，小心地取出磁带，从每尾轻轻拉。不要从尾部翻录的磁带，因为这样做可能会引起疼痛的MICE. </li><li>回国后的老鼠他们的殖民地房间丢弃的粪便勃利和尿液收集托盘和与消毒液擦拭仪器。 </li></ol> 3。行为分析<ol><li>一般来说，尖沙咀是从开始到六分钟的长度完成。不同的另一种广泛使用的抗抑郁疗效程序，强迫游泳测试，整个会议取得。这是由于一般的发现，老鼠往往体现不动早在尖沙咀。 </li><li>在我们的实验室中，我们的视频文件直接从相机上传到PC进行分析。 </li><li>在行为分析每个鼠标移动测量花费的时间。虽然它可以直接测量的不动时间，我们发现，积极运动更容易发现和措施，而不是缺乏这种运动。 </li><li>尖沙咀行为分析的最重要的方面是一致的运动识别作为真正的流动记录S。小鼠，尤其是在会议开始时，清单公然逃避相关的行为。这些措施包括试图达到的器具的墙壁和吊杆，身体的强烈震动，类似于运行的四肢运动。这些动作显然构成的流动性。这些行为，然后消退而变得微妙。在我们的实验室只限于前腿，但没有参与的后腿，不计算为流动的小运动。此外，喜欢的势头，由于波动的振荡和摆在早些时候的流动性较量的经验也不能算作流动。 </li><li>在我们的实验室中，我们使用的时间测量屏幕上的秒表软件（Xnote秒表，dnSoft研究小组）。在屏幕上使用两个独立的秒表。第一个秒表倒计时360秒和警报行为分析期结束时的观察员。第二STOpwatch控制观察员措施所花费的时间移动。某些秒表软件有能力来分配钥匙启动和停止功能，使屏幕上的键盘可以控制秒表。相反，我们使用普通键盘输入设备，俗称为"手柄"控制的秒表。 </li><li>如果有一个以上的测试，并在屏幕上的鼠标目前，它是一个好主意，掩盖了其他的动物，使他们的动作不会分散观察员。这可以通过使用其他程序窗口或通过物理覆盖与纸屏幕上的其他老鼠。 </li><li>有可能会发生一些偏差，如果流动性的时间总额为一个特定的鼠标经过前完成分析会议可见。如果使用屏幕上的秒表是，我们建议涵盖所有，但秒表毫秒小数。覆盖秒表的观察员只知道是否秒表开启或关闭在任何时候，但不知道的总时间过去了，不带任何偏见，因此不能受到影响。使用这种方法的观察员，而盲目的动物组分配，不会有一个在每个动物的流动性水平的总体思路。 </li><li>观察者可靠性测试应为每一个新的观察员进行，才开始收集数据从实验动物。在我们的实验室，每一个新的观察员第一只手表一位经验丰富的观察员，而他或她是得分。在新的观察员获得足够的信心，以区别于万马齐喑流动，然后他们在实时得分与经验丰富的观察员观看，并指出任何错误。一旦成功完成这一阶段，新的观察员将分析具体的培训目的，我们在我们的实验室保持尖沙咀视频设置。只有在一个观察者相关的高层次是与经验丰富的观察员获得的调查分析，从实际尖沙咀视频实验。我们从这些培训分析的数据进行归档，以构成一个实验室内部标准。我们已经观察到的菌株之间的差异，他们表示流动（不动）的行为，以及男女之间的时间平均不动的方式。一个新的应变，性别，或转基因小鼠模型在实验室测试时，有必要再次进行这种类型的可靠性分析。 </li></ol> 4。代表性的成果我们已经使用了尖沙咀评估抗抑郁反应，锂在不同小鼠品系的治疗（图1）3 。本实验的实验的详细资料载于能等人，2011年3。 <img src="/files/ftp_upload/3769/3769fig1.jpg" alt="图1" /> 图1:在五个近交系小鼠品系的慢性锂管理后悬尾实验不动时间。小鼠组的房子D，每笼4只。在锂治疗组小鼠接受了含有锂议员三个星期4克/公斤氯化锂。控制动物无氯化锂相同的食品。 **:P &lt;0.01，***:P &lt;0.001表示显著，配对t检验。数据表示为平均值± SEM。 ñ :10 - 12动物每组每一株。 （从图3转载）。 在这个例子中，只在C57BL/6J小鼠和DBA/2J株慢性锂食物中提供的一个显着的治疗效果观察。在其他3株，不动时间的统计上的显著减少。这些数据表明，慢性锂治疗的抗抑郁反应应变依赖也已经与其他 4-8抗抑郁药物观察。

<ol>
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L. e. a. r. y., F, O., Lucki, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+tests+for+measuring+the+effects+of+antidepressants+in+mice.">Automated tests for measuring the effects of antidepressants in mice.</a> Pharmacology Biochemistry and Behavior. 78, 269-274 (2004).</li><li>Gould, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+AMPA+receptors+in+the+antidepressant-like+effects+of+lithium+in+the+mouse+tail+suspension+test+and+forced+swim+test.">Involvement of AMPA receptors in the antidepressant-like effects of lithium in the mouse tail suspension test and forced swim test.</a> Neuropharmacology. 54, 577-587 (2008).</li><li>Gould, T. D., Dao, D. T., Kovacsics, C. E., Gould, T. 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作者宣称没有利益冲突。

Steru 等尖沙咀发展。影响以前开发的强迫游泳测试10-12。类似强迫游泳测试，在尖沙咀的小鼠，被放置在一个无法回避的，但适度紧张的情况。缺乏逃生相关的行为被认为是静止的。强迫游泳测试一样，尖沙咀是最好的抗抑郁药物疗效的评价的验证测试，但也可以用来评估环境，神经生物学和基因操纵13-18的影响。在强迫游泳测试，在尖沙咀有没有体温过低的危险，?...

<ul><li>磁带</li><li>定时器</li><li>摄像机</li><li>白噪声发生器（可选） </li><li> Climbstoppers（可选，取决于使用时应变） </li></ul>

the tail suspension test

尾部悬吊试验是鼠标行为潜在的抗抑郁药物的筛选有用的测试，并评估预期影响抑郁症有关的行为的其他操作。小鼠暂停它们的尾巴，用胶带，在这样的位置，无法逃避或持有到附近的表面。在测试过程中，通常是6分钟，造成逃生导向的行为进行量化。尾部悬吊的测试是一个准抗抑郁化合物的高通量筛选药物发现有价值的工具。在这里，我们描述了实施这种潜在的可能出现的问题，以及如何避免它们进一步强调测试所需的细节。我们还提供了一个尾部的攀爬行为的解决方案，如广泛使用的C57BL / 6的一个共同的问题，使这个测试在某些小鼠品系的无用。具体来说，我们通过一个小塑料鼠标尾巴，防止尾登山行为缸中止之前。最后，我们详细介绍如何手动评分，在这个测试中表现的行为。

Watch this Scientific Journal Video about The Tail Suspension Test at JoVE.com

The Tail Suspension Test

尾部悬吊试验验证实验程序，以评估对小鼠的抗抑郁药物治疗的疗效。小鼠暂停六分钟，它们的尾巴逃生相关的行为进行评估。我们描述进行悬尾实验中使用的程序。

悬尾实验

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

Research

JoVE Journal

Neuroscience

78.9K 次观看.  University of Maryland School of Medicine. 尾部悬吊试验验证实验程序，以评估对小鼠的抗抑郁药物治疗的疗效。小鼠暂停六分钟，它们的尾巴逃生相关的行为进行评估。我们描述进行悬尾实验中使用的程序。

视频: The Tail Suspension Test

One Dimensional Turing-Like Handshake Test for Motor Intelligence

In the Turing test, a computer model is deemed to "think intelligently" if it can generate answers that are not distinguishable from those of a human. However, this test is limited to the linguistic aspects of machine intelligence. A salient function of the brain is the control of movement, and the movement of the human hand is a sophisticated demonstration of this function. Therefore, we propose a Turing-like handshake test, for machine motor intelligence. We administer the test through a telerobotic system in which the interrogator is engaged in a task of holding a robotic stylus and interacting with another party (human or artificial). Instead of asking the interrogator whether the other party is a person or a computer program, we employ a two-alternative forced choice method and ask which of two systems is more human-like. We extract a quantitative grade for each model according to its resemblance to the human handshake motion and name it "Model Human-Likeness Grade" (MHLG). We present three methods to estimate the MHLG. (i) By calculating the proportion of subjects' answers that the model is more human-like than the human; (ii) By comparing two weighted sums of human and model handshakes we fit a psychometric curve and extract the point of subjective equality (PSE); (iii) By comparing a given model with a weighted sum of human and random signal, we fit a psychometric curve to the answers of the interrogator and extract the PSE for the weight of the human in the weighted sum. Altogether, we provide a protocol to test computational models of the human handshake. We believe that building a model is a necessary step in understanding any phenomenon and, in this case, in understanding the neural mechanisms responsible for the generation of the human handshake.

We present a Turing-like Handshake test administered through a telerobotic system in which the interrogator is holding a robotic stylus and interacting with another party (human or artificial). We use a forced choice method, and extract a measure for the similarity of the artificial model to a human handshake.

一维类似图灵汽车情报握手测试

In the Turing test, a computer model is deemed to "think intelligently" if it can generate answers that are not distinguishable from those of a human.  ...

科研

神经科学

The Mouse Forced Swim Test

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

小鼠强迫游泳实验

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative to understand the cellular and molecular changes involved in maintaining and repairing NMJs. For this purpose, we have developed a method to reliably and consistently examine regenerating NMJs in mice. This nerve injury method involves crushing the common fibular nerve as it passes over the lateral head of the gastrocnemius muscle tendon near the knee. Using 70 day old female mice, we demonstrate that motor axons begin to reinnervate previous postsynaptic targets within 7 days post-crush. They completely reoccupy their previous synaptic areas by 12 days. To determine the reliability of this injury method, we compared reinnervation rates between individual 70 day old female mice. We found that the number of reinnervated postsynaptic sites was similar between mice at 7, 9, and 12 days post-crush. To determine if this injury assay can also be used to compare molecular changes in muscles, we examined levels of the gamma-subunit of the muscle nicotinic receptor (gamma-AChR) and the muscle-specific kinase (MuSK). The gamma-AChR subunit and MuSK to are highly upregulated following denervation and return to normal levels following reinnervation of NMJs. We found a close relationship between transcript levels for these genes and innervation status of muscles. We believe that this method will accelerate our understanding of the cellular and molecular changes involved in repairing the NMJ and other synapses.

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

腓神经损伤方法:可靠检测，以确定和测试因素修复神经肌肉接头

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

漫反射光谱分析: 在拇指下进行毛细管填充试验

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing factor. Single-point sampling measures are inadequate to fully understand the biological significance of the secretory pattern of pulsatile hormones either under normal physiologic conditions or during conditions of dysregulation. Luteinizing hormone (LH) is synthesized by the anterior pituitary gonadotrope&#160;cells and secreted in a pulsatile pattern which requires frequent collection of blood samples for pulse assessment. This has not been possible in mice until recently, due to the development of a high-sensitivity LH assay and advancement in a technique for frequent low-volume sample collection, initially described by Steyn and colleagues.1 Here we describe a protocol for the frequent peripheral blood sample collection from mice with sufficient handling acclimatization to detect pulsatile secretion of LH. The current protocol details an expanded acclimatization period that allows assessment of robust and continuous pulses of LH over multiple hours. In this protocol, the tip of the tail is clipped&#160;and blood is collected from the tail using a hand-held pipette. For assessment of pulsatile LH in gonadectomized mice, serial samples are collected every 5-6 min for 90-180 min. Importantly, the collection of blood and measurement of robust pulses of LH can be accomplished in awake, freely behaving mice, given adequate handling acclimatization and effort to minimize environmental stressors. Sufficient acclimatization can be achieved within 4-5 weeks prior to blood collection. This protocol highlights advances in the methodology to ensure collection of whole blood samples for assessment of pulsatile LH secretion patterns over multiple hours in the mouse, a powerful animal model for neuroendocrine research.

Here we present a tail-tip blood sampling protocol for frequent sample collection in unrestrained mice. This method is useful for assessing patterns of pulsatile luteinizing hormone secretion and could be adapted for analysis of other circulating factors.

小鼠经常尾端采血对脉动性促黄体激素分泌的评价

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

创伤性脑损伤后评估成年大鼠空间工作记忆的指标测试

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...

同时记录脑电图的大鼠脑脊液和血液

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

作者聚焦:获取高质量的 CSF 和血液样本以发现癫痫生物标志物 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

在脑电图记录期间从大鼠的侧尾静脉采集脑脊液和血液

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

量化 果蝇 神经系统基因的 Poly（A） 长度

Measurement of Poly A Tail Length from Drosophila Larva Brain and Cell Line

Polyadenylation is a crucial posttranscriptional modification that adds poly(A) tails to the 3' end of mRNA molecules. The length of the poly(A) tail is tightly regulated by cellular processes. Dysregulation of mRNA polyadenylation has been associated with abnormal gene expression and various diseases, including cancer, neurological disorders, and developmental abnormalities. Therefore, comprehending the dynamics of polyadenylation is vital for unraveling the complexities of mRNA processing and posttranscriptional gene regulation. 
This paper presents a method for measuring poly(A) tail lengths in RNA samples isolated from Drosophila larval brains and Drosophila Schneider S2 cells. We employed the guanosine/inosine (G/I) tailing approach, which involves the enzymatic addition of G/I residues at the 3' end of mRNA using yeast poly(A) polymerase. This modification protects the RNA's 3' end from enzymatic degradation. The protected full-length poly(A) tails are then reverse-transcribed using a universal antisense primer. Subsequently, PCR amplification is performed using a gene-specific oligo that targets the gene of interest, along with a universal sequence oligo used for reverse transcription. 
This generates PCR products encompassing the poly(A) tails of the gene of interest. Since polyadenylation is not a uniform modification and results in tails of varying lengths, the PCR products display a range of sizes, leading to a smear pattern on agarose gel. Finally, the PCR products are subjected to high-resolution capillary gel electrophoresis, followed by quantification using the sizes of the poly(A) PCR products and the gene-specific PCR product. This technique offers a straightforward and reliable tool for analyzing poly(A) tail lengths, enabling us to gain deeper insights into the intricate mechanisms governing mRNA regulation.

作者聚焦:使用 Poly A Tail 分析技术探索 mRNA 研究的前沿

The protocol describes an efficient and reliable method for quantifying the poly(A) length of the gene of interest from the Drosophila nervous system, which can be easily adapted to tissues or cell types from other species.

果 蝇 幼虫脑和细胞系的Poly A尾长测量

Polyadenylation is a crucial posttranscriptional modification that adds poly(A) tails to the 3' end of mRNA molecules. The length of the poly(A) tail is ...