This study has been supported by the grant NIHM R01 MH091816 and R21 MH084043 to TDG.

1. Materials 
1. Suspension Box
The tail-suspension test (TST) involves suspending mice above the ground by their tails. At the most basic level, the procedure only requires a suspension bar or shelf ledge, and tape. However, the experimenter should consider the use of a background that provides optimal contrast. Additionally, it is prudent to take steps to prevent mice from observing other animals that are being tested. In our laboratory, we use specially manufactured tail suspension boxes (Four-Hour Day, Baltimore MD), made of plastic with the dimensions (55 height X 60 width X 11.5 cm depth). In order to prevent animals from observing or interacting each other, each mouse is suspended within its own three-walled rectangular compartment (55 height X 15 width X 11.5 cm depth). The mouse is suspended in the middle of this compartment and the width and depth are sufficiently sized so that the mouse cannot make contact with the walls. In this setting, the approximate distance between the mouse's nose and the apparatus floor is 20-25 cm. There are four such identical compartments in the apparatus allowing us to test four mice at a time. An aluminum suspension bar (1 cm. height X 1 cm. width x 60 cm. length), used to suspend the tail of each mouse, is positioned on the top of the box. The dimensions that we use in our laboratory should be regarded as a general reference. For example, the size of individual compartments of the tail-suspension box can be increased if a large outbred mouse strain (e.g. CD-1) is used.
At the bottom of each compartment we place a detachable aluminum tray that collects feces or urine from the animals. We use a dark grey box for albino animals and a cream colored box for mice of other coat colors. This arrangement gives us better contrast and therefore more reliable behavioral scoring of the test.
2. Tape
Tape should adhere securely to both the mouse&#x2019;s tail and the suspension bar, and be strong enough to carry the weight of the mouse being tested (e.g. TimeMed Labeling Systems, Inc, Burr Ridge IL, 1.9 cm width). However, the tape should not be excessively sticky since at the end of the session it will be removed from the tail. We cut 17 cm fragments of tape and place a mark 2 cm from one end. This 2 cm portion was used for attaching the tape to the tail, while the remaining 15 cm was used for suspension of the mouse.
3. Timer



4. Video recording device
A video camera and a tripod (or other support structure) are needed. Since this test usually involves multiple animals being tested at the same time, live scoring will be difficult and is not advised. The video camera should record in high enough resolution to render a quality picture that will be used later for behavioral scoring. Always make certain that there is sufficient recording memory in the camera before starting the test. We use a video camera that records digitally without the use of physical media (i.e. video cassette), allowing for straightforward transfer of videos. 
5. White noise generator
The noise generator should mask intermittent environmental sounds. The use of a noise generator is particularly recommended in laboratory environments where sudden loud noises can be heard that would potentially startle mice. In our experimental room the ambient noise level (without the white noise generator activated) is around 60 dB. The total sound level with the white noise generator activated at the location where the TST is conducted is 70-72 dB. However, it should be noted that these figures are provided as example only, and each laboratory should select the right noise levels according to their unique environment and circumstances.
6. Cleaning supplies
The suspension box should be wiped thoroughly after each session with a sterilization solution (e.g. MB-10, Quip Laboratories Inc., Wilmington, DE, or similar).
7. Climbstoppers (optional: dependent upon strain used)
Mice of some backgrounds, such as C57BL/6, may climb their tails during the test1. Clear hollow cylinders (4 cm length, 1.6 cm outside diameter, 1.3 cm inside diameter, 1.5 grams) that are cut to four cm length (by FourHourDay Inc, Baltimore MD) from polycarbonate tubing (#8585K41, McMaster-Carr, Santa Fe Springs, CA) are placed around the tails of mice to prevent such tail climbing behavior2,3. These devices can be made in any laboratory with common tools and materials. 
2. Behavioral Procedures
The overall experimental design should reflect proper counterbalancing between variables specific to your experiment. For example, in our experiments we try to represent each group equally in every TST session (i.e., if there are four treatment groups, each will be represented in each session). Also, the positions of mice are rotated in a way such that mice from each treatment group are placed in a different position in each session.
<ol>
 <li>Place the camera in position. The camera should be as close as possible in order to obtain the highest possible resolution of the animals.</li>
 <li>Tape fragments that will be used during the session should be cut, marked, and prepared for the session.</li>
 <li>Start the white noise generator, if being used, before the mice are introduced to the testing room. The level of white noise should only be enough to mask external noises. Avoid a high volume and make sure the same level of white noise is used for all animals.</li>
 <li>Bring the animals into the testing room. If the room where the animals reside and the testing room are adjacent to each other with similar ambient conditions, no acclimation period may be necessary. Otherwise, place the animals in the testing room for a period of acclimation (generally at least one hour). Be aware that the other animals placed in the same room can sense olfactory and ultrasonic cues.</li>
 <li>If a strain known to climb their tails, such as C57BL6/J, is being used place Climbstoppers around the tails prior to applying the tape.</li>
 <li>Adhere the tape to the tails of mice. Use the marked end of the tape and make sure the tape sticks to the tail and back on itself. The tape should be applied to the very end of the tail with 2-3 millimeters of tail remaining outside of the tape. </li>
 <li>Once each piece of tape is attached to a mouse tail stick the middle portion of the tape to the inner walls of the cage. This will help prevent tangling of tape pieces to each other. Note that confinement in this fashion may cause stress and that it is important to complete the process of applying the tape to each animal as soon as possible.</li>
 <li>Once all tape is applied, start the recording and identify the session before the mice are suspended.</li>
 <li>Suspend the animals by placing the free end of the tape on the suspension bar or shelf in an order that is counterbalanced between treatment groups. Suspend mice in a way that is does not obstruct the camera view because the entire TST session will be scored and obstructing the camera will lead to the inability to assess behaviors during that time. Check that all tape ends are hanging parallel to the suspension bar or shelf with a similar length of slack for each mouse. </li>
 <li>At the end of the session (which is typically six minutes), return the animals to their homecage and carefully remove the tape from each tail by gently pulling it off. Do not rip the tape from the tail since doing so may cause pain to the mice. </li>
<li>After returning the mice to their colony room discard the fecal boli and urine from the collection trays and wipe the apparatus with a sterilizing solution. </li>
</ol>
3. Behavior Analysis
<ol>
 <li>Generally, the TST is from start to finish six minutes in length. Unlike another widely used antidepressant efficacy procedure, the forced swim test, the whole session is scored. This is due to the general finding that mice tend to manifest immobility earlier in the TST.</li>
 <li>In our laboratory we upload the video files directly from the camera to a PC where the analysis is conducted.</li>
 <li>During the behavioral analysis the time that each mouse spends as mobile is measured. While it is possible to measure the immobility time directly, we have found it easier to detect and measure active movements rather than the lack of such movements. </li>
 <li>The most important aspect of TST behavioral analysis is the consistent identification of movements that are recorded as bona fide mobility. Mice, especially at the beginning of the session, manifest behaviors that are overtly escape related. These include trying to reach the walls of the apparatus and the suspension bar, strong shaking of the body, and movement of the limbs akin to running. These movements clearly constitute mobility. These behaviors then subside and become subtler. In our laboratory small movements that are confined to the front legs but without the involvement of the hind legs are not counted as mobility. Additionally, oscillations and pendulum like swings that are due to the momentum gained during the earlier mobility bouts also are not counted as mobility.</li>
 <li>In our laboratory we use an on-screen stopwatch software for time measurements (Xnote Stopwatch, dnSoft Research Group). Two separate stopwatches are used on the screen. The first stopwatch counts down from 360 seconds and alerts the observer when the behavioral analysis period ends. The second stopwatch &mdash;controlled by the observer&#x2014;measures the time spent mobile. Certain stopwatch software has the ability to assign keys to start and stop functions, so that on-screen the keyboard can control the stopwatch. Instead of a regular keyboard, we use an input device commonly known as a 'gamepad' to control the stopwatches. </li>
 <li>If there is more than one mouse tested and present on the screen it is a good idea to cover the other animals so that their movements will not distract the observer. This can be accomplished by using another program window or by physically covering the other mice on the screen with paper.</li>
 <li>There may be some bias occurring if the total amount of mobility time elapsed for a particular mouse is visible prior to completion of the analysis session. If an on-screen stopwatch is used, we suggest to cover all but the millisecond decimals of the stopwatch. By covering the stopwatch the observer only knows whether the stopwatch is on or off at any point but does not know the total time elapsed and therefore cannot be affected by any bias. Using this approach the observer, while blind to the group assignments of the animals, will not have a general idea of level of mobility in each animal.</li>
 <li>An interobserver reliability test should be conducted for every new observer before beginning to collect data from test animals. In our laboratory each new observer first watches an experienced observer while he or she is scoring. After the new observers gain enough confidence to differentiate mobility from immobility, they then score in real time with the experienced observer watching and pointing out any mistakes. Once this phase is successfully completed, the new observers will analyze a specific set of TST videos that we maintain in our laboratory for training purposes. Only after a high level of interobserver correlation is obtained with the experienced observer does an investigator analyze TST videos from actual experiments. We archive the data from these training analyses to constitute an internal standard for the laboratory. We have observed differences between strains in the manner in which they express mobility (and immobility) behaviors, and mean immobility time between sexes. When a new strain, sex, or genetically modified mouse model is tested in the laboratory it is necessary to again undertake this type of reliability analysis. </li>
</ol>
4. Representative Results
We have used the TST to assess antidepressant-like responses to lithium treatment in various mouse strains (Figure 1)3. Experimental details of this experiment are published in Can et al., 20113. 
<img src="/files/ftp_upload/3769/3769fig1.jpg" alt="Figure 1" /> 
Figure 1. Immobility time in the tail suspension test after chronic lithium administration in five inbred mouse strains. Mice were group housed, four mice per cage. Mice in the lithium treatment group received lithium chow containing 4 g/kg LiCl for three weeks. Control animals received identical food without the lithium chloride. **:p&lt;0.01, ***:p&lt;0.001 denote a significant, unpaired t-test. Data are expressed as mean &plusmn; SEM. n:10-12 animals per group for each strain. (figure reproduced from 3).
In this example, a significant treatment effect of chronic lithium delivered in food is observed only in C57BL/6J and DBA/2J strains. In the other three strains, no statistically significant decrease in immobility time was observed. These data indicate that an antidepressant-like response to chronic lithium treatment is strain dependent as has also been observed with other antidepressant medications4-8.

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Authors declare no conflicts of interest.

Development of the TST by Steru et al.9 was influenced by the previously developed forced swim test10-12. Similar to the forced swim test, in the TST mice are placed in an inescapable but moderately stressful situation. Lack of escape related behavior is considered immobility. Like the forced swim test, the TST is a test best validated for the evaluation of antidepressant efficacy of drugs, but also used to evaluate the effects of environmental, neurobiological, and genetic manipulations<su...

<ul>
	<li>Tape</li>
	<li>Timer</li>
	<li>Video Camera</li>
	<li>White Noise Generator (optional)</li>
	<li>Climbstoppers (optional; depending upon strain used)</li>
</ul>

the tail suspension test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

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

The Tail Suspension Test

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

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

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79.1K Views.  University of Maryland School of Medicine. The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

Video: 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.

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.  ...

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.

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.

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.

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One&#039;s Thumb

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.

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.

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.

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

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

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.

Author Spotlight: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery 

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.

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

 Quantifying the Poly(A) Length of Genes from the &lt;em&gt;Drosophila&lt;/em&gt; Nervous System

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.

Author Spotlight: Exploring the Frontier of mRNA Research with Poly A Tail Analysis Techniques

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.

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 ...

Collection of Blood from the Tail Vein of Rats During EEG Recordings

To begin, coat a butterfly needle and its tubing with 1%potassium EDTA solution. Cut the tubing just behind the needle in order to collect blood dropwise. Place an anesthetized rat on the stereotaxic frame while maintaining anesthesia through a face mask.Put a heating pad under the animal, keeping a part of its tail in direct contact with the pad. Move the animals back such that it faces sideways, and the lateral tail vein is visible at the top. Dip the tail in warm water to dilate the lateral vein.Then wipe the tail with 70%ethanol. Put warm light onto the tail using a regular incandescent bulb. Insert the 21G butterfly needle into the lateral tail vein at an angle of 20 degrees to a depth of five millimeters.Next, collect blood in a 500 microliter vacuum collection tube containing five milligrams of potassium EDTA as an anticoagulant. Remove the needle and stop the flow of the blood by putting pressure on the puncture site. Return the rat to its home cage.Next, gently invert the tube 10 times to mix anticoagulant in the blood. Make 1.5 centimeter deep holes in the ice and place the tubes in them vertically. Within one hour of collection, centrifuge the blood sample in a refrigerated centrifuge to separate the plasma.Then aspirate about 200 microliters of plasma, avoiding the red and white blood cell layer. Place the withdrawn plasma into a 0.2 milliliter sterile microtube. Put aside five microliters of the sample for quality control and store the remaining sample at minus 80 degrees Celsius until analysis.Blood was withdrawn from rats at five time points using different methods, such as the vacuum technique on day 52, tail milking on day 55, and drop techniques on days 58 to 64. The quality of plasma samples evaluated using UV spectrophotometry showed that the plasma was pink-colored with a mean absorbance value of 0.647 when using the vacuum technique. Similarly, the tail milking technique resulted in pink-colored plasma with mean absorbance of 0.620.In contrast, with the gravity-enabled drop withdrawal system, clear plasma samples were obtained with significantly reduced mean plasma absorbance values, suggesting that this procedure is optimal for getting high quality samples for analyses.

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...