This work has been funded, in part, by the Canada Foundation for Innovation.

1. System Preparation
<ol>
	<li>Clear the capture volume of any reflective material that may be observed by the cameras. This decreases the chances of actual skin-based markers being confused with stationary background markers during the movement testing and improves the overall accuracy of the session.</li>
	<li>Calibrate the cameras by aiming all cameras on stationary markers at fixed positions within the laboratory. Extend the static calibration to dynamic movements using moving markers placed at known distances. Be sure to cover as much of the capture volume as possible to optimize the calibration.</li>
	<li>Organize all materials (reflective markers, measurement devices, etc.) to be used for patient preparation. This improves efficiency during testing and reduces patient burden.</li>
</ol>
2. Patient Preparation
<ol>
	<li>Expose as much skin as possible over the joints and body segments intended to be measured. Minimize the amount of loose fitting clothing and ensure that any pieces of clothing that may interfere with the ability of the cameras to visualize the reflective markers are constrained. This can be done using tape or clips. Whenever possible, ensure that markers are affixed directly to the skin.</li>
	<li>Prepare the skin for marker fixation. Shaving or abrading the area may be necessary in instances where hair is present or when the skin surface is excessively sweaty or oily. Wiping the area clear using rubbing alcohol or a similar liquid can be useful. These steps are important to maximize adherence between the marker and the skin, and to prevent markers from falling off.</li>
	<li>Palpate for key anatomical landmarks based on the marker set to be used. Marking the skin at the actual landmark will improve accuracy for marker placement and provide information necessary in cases of markers falling off during assessment.</li>
	<li>Affix reflective markers over the anatomical landmarks according to the specifications of the marker set. Most marker sets will include a minimum of 12-15 markers placed bilaterally over the lower limbs and various anatomical landmarks of the upper body. It is important to note that the ability to re-create actual skeletal movement will depend on the positioning of skin-based markers. As such, careful consideration must be made when determining the biomechanical model to be used.</li>
	<li>Take measurements for important anthropometric data, if required. Depending on the biomechanical model, these data may be needed to calculate segment lengths, positions of joint centers of rotation, and overall inertial properties of the moving segments and limbs during offline processing of biomechanics data.</li>
</ol>
3. Motion Analysis and Delivery of Real-time Feedback
<ol>
	<li>Have the subject stand in the middle of the capture volume for an initial static trial lasting approximately 3 sec. This trial is necessary to ensure that all relevant markers are visible and to calculate segment orientations.</li>
	<li>Using the data collection software, label all markers as appropriate and create a template specific to the anthropometric characteristics of the individual. Matching marker placement to the individual body size will improve the real-time tracking and analysis of data. It is especially important to create a model of movement that can incorporate redundancies of marker positioning. In instances where marker occlusion or drop-off occurs, the ability to utilize additional marker positions where appropriate to produce the appropriate kinematic characteristic and maintain real-time display without breaks in the data.</li>
	<li>Perform an initial motion analysis trial lasting from 10-30 sec. This is required to obtain baseline data and can also be used as the first mechanism of providing feedback of results to the patient. Consultation with the patient regarding relevant findings is important to assist in the motor learning required when producing new movement patterns.</li>
	<li>Have the therapist explain the purpose of the intended movement modification. This should include both biomechanical and clinical rationales for the modification and how it is unique to the given pathology. Demonstration of the movement modification by the therapist will enhance motor learning by the patient. The movement modification will typically be determined based on the biomechanical and clinical presentation of the patient during treatment, or the research question to be examined if solely for research purposes.</li>
	<li>Begin the movement retraining session. If using a treadmill, allow the subject to choose their own preferred speed and provide a couple of minutes to reach a steady-state. This also allows the patient to become familiar and comfortable with the equipment, experimental set-up, and protocol.</li>
	<li>Provide feedback to the patient during performance of the movement. This can take the form of many different approaches, and combination of these is beneficial during early training. Start with less technical methods such as verbal feedback and progress to real-time biofeedback. Utilization of real-time biofeedback should always include clear display of a maximum of one outcome variable at a time.</li>
	<li>Provide sufficient time for the patient to practice the new movement. Effective motor learning is not achieved instantaneously. Instead, constant practice of the new movement characteristics will assist in ensuring re-formulation of the motor program responsible for that movement. A typical retraining intervention may require 8-10 focused training sessions, each lasting between 30 and 60 min.</li>
</ol>
4. Patient De-briefing and Subsequent Training Sessions
<ol>
	<li>Discuss the important findings and outcomes of the session with the patient. Important factors to focus on should include variability in performance, adherence to the prescribed movement modification and further description of the rationale and importance of the modification.</li>
	<li>Obtain input regarding the session from the patient. Given that each patient's preferences will likely differ, it may be necessary to modify the delivery of the intervention for a given individual. These should be identified early to optimize effectiveness.</li>
	<li>Determine the plan for subsequent training sessions, if necessary. If a multi-session intervention is chosen, subsequent training sessions should use a faded feedback approach to enhance motor learning. Provide less overall feedback and alternate between time blocks of feedback and no feedback in future sessions.</li>
</ol>

<ol>
	<li>Ivanenko, Y. P., Poppele, R. E., Lacquaniti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+control+programs+and+walking.">Motor control programs and walking.</a> Neuroscientist. 12, 339-348 (2006).</li><li>Woodford, H., Price, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMG+biofeedback+to+improve+lower+extremity+function+after+stroke.">EMG biofeedback to improve lower extremity function after stroke.</a> Cochrane Database Syst. Rev. 2007, CD004585 (2007).</li><li>Moreland, J. D., Thomson, M. A., Fuoco, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromyographic+feedback+to+improve+lower+extremity+function+after+stroke:+a+meta-analysis.">Electromyographic feedback to improve lower extremity function after stroke: a meta-analysis.</a> Arch. Phys. Med. Rehabil. 79, 134-140 (1998).</li><li>Colborne, G. R., Wright, F. V., naumann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+of+triceps+surae+EMG+in+gait+of+children+with+cerebral+palsy:+a+controlled+study.">Feedback of triceps surae EMG in gait of children with cerebral palsy: a controlled study.</a> Arch. Phys. Med. Rehabil. 75, 40-45 (1994).</li><li>Binder, S. A., Moll, C. B., Wolf, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+electromyographic+biofeedback+as+an+adjunct+to+therapeutic+exercise+in+treating+the+lower+extremities+of+hemiplegic+patients.">Evaluation of electromyographic biofeedback as an adjunct to therapeutic exercise in treating the lower extremities of hemiplegic patients.</a> Phys. Ther. 61, 886-893 (1981).</li><li>Tate, J. C., Milner, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+kinematic,+temporospatial,+and+kinetic+biofeedback+during+gait+retraining+in+patients:+a+systematic+review.">Real-time kinematic, temporospatial, and kinetic biofeedback during gait retraining in patients: a systematic review.</a> Phys. Ther. 90, 1123-1134 (2010).</li><li>Miyazaki, T., Wada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+load+at+baseline+can+predict+radiographic+disease+progression+in+medial+compartment+knee+osteoarthritis.">Dynamic load at baseline can predict radiographic disease progression in medial compartment knee osteoarthritis.</a> Ann. Rheum. Dis. 61, 617-622 (2002).</li><li>Barrios, J., Crossley, K., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+retraining+to+reduce+the+knee+adduction+moment+through+real-time+visual+feedback+of+dynamic+knee+alignment.">Gait retraining to reduce the knee adduction moment through real-time visual feedback of dynamic knee alignment.</a> J. Biomech. 43, 2208-2213 (2010).</li><li>Hunt, M. A., Simic, M., Hinman, R. S., Bennell, K. L., Wrigley, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+a+gait+retraining+strategy+for+reducing+knee+joint+loading:+Increased+trunk+lean+guided+by+real-time+biofeedback.">Feasibility of a gait retraining strategy for reducing knee joint loading: Increased trunk lean guided by real-time biofeedback.</a> J. Biomech. 44, 943-947 (2011).</li><li>Simic, M., Hunt, M. A., Bennell, K. L., Hinman, R. S., Wrigley, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trunk+lean+gait+modification+and+knee+joint+load+in+people+with+medial+knee+osteoarthritis:+The+effect+of+varying+trunk+lean+angles.">Trunk lean gait modification and knee joint load in people with medial knee osteoarthritis: The effect of varying trunk lean angles.</a> Arthritis Care Res. , (2012).</li><li>Hunt, M. A., Simic, M., Hinman, R. S., Bennell, K. L., Wrigley, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+a+gait+retraining+strategy+for+reducing+knee+joint+loading:+Increased+trunk+lean+guided+by+real-time+biofeedback.">Feasibility of a gait retraining strategy for reducing knee joint loading: Increased trunk lean guided by real-time biofeedback.</a> J. Biomech. , (2010).</li><li>Mundermann, A., Asay, J., Mundermann, L., Andriacchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+increased+medio-lateral+trunk+sway+for+ambulatory+mechanics.">Implications of increased medio-lateral trunk sway for ambulatory mechanics.</a> J. Biomech. 41, 165-170 (2008).</li></ol>

No conflicts of interest declared.

Real-time feedback of performance during movements such as walking can be a valuable adjunct to standard motion analysis approaches. Though in its relative infancy, research into specific and discrete movement modifications will certainly benefit from the ability to produce the desired modification with accuracy and in real-time. For example, if the patient requires a specific amount of movement modification, this amount can be measured and provided during the actual movement. The approach presented here can be used to t...

Any significant change to the neuromuscular or musculoskeletal structure of the lower limb will likely have an impact on the characteristics of movement and associated physical function. Accordingly, improvement in physical function is an important outcome of any rehabilitation intervention. Normal repetitive movements such as walking are generally governed by motor programs that contain the necessary control information needed to activate muscles with the correct intensity and timing1. These motor programs are necessary to improve the automaticity of movement, thus reducing the amount of control devoted to movement and permitting attention to be paid to other higher level tasks. However, given the role of motor programs in movement and the fact that these programs are refined over a number of years, changing movement performance after injury or disease is a challenging venture.
Traditionally, movement retraining interventions have been predicated on providing sufficient feedback of movement performance to ensure that the new information is incorporated into the new and evolving motor program. Simple, yet effective, approaches include verbal feedback with global instructions (e.g. "bend more", "keep your knee straight") as well as mechanisms of providing visual feedback such as use of a mirror or video recording devices. Though these indirect strategies are useful, especially in clinical settings with limited resources, they are limited by their difficulty in providing discrete and quantifiable measures of movement variables. As a result, supplementing these techniques with additional more direct methods of feedback will likely enhance the motor re-learning desired.
There is much acceptance in the research and clinical communities that providing feedback of discrete, quantifiable outcomes of movement characteristics can improve performance during a movement retraining intervention. For example, instantaneous visual or auditory feedback of muscle activation intensity using electromyographic biofeedback devices has become a mainstay in the rehabilitation of movement, particularly in people with stroke2-3, cerebral palsy4, or chronic hemiplegia5. In contrast, feedback of movement kinematics (joint and segment angles) has proven to be less utilized due to a difficulty in assessing and measuring these outcomes quickly and accurately. Indeed, though quantitative, laboratory-based analysis of motion features prominently in biomechanics research and has begun to be incorporated into the clinical setting, the vast majority of motion analysis usage is reserved for offline analysis after testing. However, there is an increasing number of studies in the literature that are using new technologies to provide feedback of gait measures as a means of improving the effectiveness of movement retraining6.
One pathology that is currently being investigated for usage of real-time biofeedback capabilities integrated with standard motion analysis systems is knee osteoarthritis (OA). Recent studies have utilized real-time feedback of gait kinematics designed specifically to reduce the load passing through the knee joint, quantified using the external knee adduction moment - a recognized risk factor for OA progression7. For example, studies have utilized real-time biofeedback of magnitudes of thigh angle8 or trunk angle9-10. Hunt et al11 provided a real-time display of trunk angle in front of participants during walking trials and showed the ability to increase exhibited trunk lean within a single training session, with accompanied reductions in knee adduction moment magnitudes. In contrast, Barrios et al8 conducted an eight-session gait retraining intervention focused on modifying dynamic frontal plane knee angle during stance and showed significant reductions in knee adduction moment values after the one-month intervention compared to baseline. These studies, and similar studies, have relied upon the ability to measure, analyze, and display the variable of interest to the patient on a continual basis. This burgeoning area of research has clinical implications for patients with a variety of pathologies that impact movement characteristics. Using examples of kinematic alterations relevant to osteoarthritis (OA) of the knee, the purpose of this paper is to describe methods required to conduct a movement retraining intervention using real-time biofeedback of walking performance.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Reflective markers</td>
 <td>3x3 Design</td>
 <td></td>
 <td>12 mm diameter</td>
 </tr>
 <tr>
 <td>Marker tape discs</td>
 <td>Discount Disposables</td>
 <td>TD-22 Electrode Collar, 8 mm</td>
 <td>Designed usage is as electrode collars</td>
 </tr>
 <tr>
 <td>Motion analysis cameras</td>
 <td>Motion Analysis Corporation</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Biofeedtrak</td>
 <td>Motion Analysis Corporation</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Matlab</td>
 <td>The Mathworks</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

movement retraining using real-time feedback of performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

An example from a single movement retraining session focusing on increased lateral trunk lean angle in a patient with knee OA is shown in Figure 2. After approximately 15 min of training using a combination of verbal and mirror-based feedback of performance, the patient was provided with real-time data pertaining to the amount of lateral trunk flexion. Training with this method continued for an additional 10 min. During normal (unmodified) trials, the patient exhibited a self-selected amount of lateral t...

Watch this Scientific Journal Video about Movement Retraining using Real-time Feedback of Performance at JoVE.com

Movement Retraining using Real-time Feedback of Performance

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

movement-retraining-using-real-time-feedback-of-performance

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13.2K Views.  University of British Columbia. Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Video: Movement Retraining using Real-time Feedback of Performance

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Real-time fMRI Biofeedback Targeting the Orbitofrontal Cortex for Contamination Anxiety

We present a method for training subjects to control activity in a region of their orbitofrontal cortex associated with contamination anxiety using biofeedback of real-time functional magnetic resonance imaging (rt-fMRI) data. Increased activity of this region is seen in relationship with contamination anxiety both in control subjects1 and in individuals with obsessive-compulsive disorder (OCD),2 a relatively common and often debilitating psychiatric disorder involving contamination anxiety. Although many brain regions have been implicated in OCD, abnormality in the orbitofrontal cortex (OFC) is one of the most consistent findings.3, 4 Furthermore, hyperactivity in the OFC has been found to correlate with OCD symptom severity5 and decreases in hyperactivity in this region have been reported to correlate with decreased symptom severity.6 Therefore, the ability to control this brain area may translate into clinical improvements in obsessive-compulsive symptoms including contamination anxiety. Biofeedback of rt-fMRI data is a new technique in which the temporal pattern of activity in a specific region (or associated with a specific distributed pattern of brain activity) in a subject's brain is provided as a feedback signal to the subject. Recent reports indicate that people are able to develop control over the activity of specific brain areas when provided with rt-fMRI biofeedback.7-12 In particular, several studies using this technique to target brain areas involved in emotion processing have reported success in training subjects to control these regions.13-18 In several cases, rt-fMRI biofeedback training has been reported to induce cognitive, emotional, or clinical changes in subjects.8, 9, 13, 19 Here we illustrate this technique as applied to the treatment of contamination anxiety in healthy subjects. This biofeedback intervention will be a valuable basic research tool: it allows researchers to perturb brain function, measure the resulting changes in brain dynamics and relate those to changes in contamination anxiety or other behavioral measures. In addition, the establishment of this method serves as a first step towards the investigation of fMRI-based biofeedback as a therapeutic intervention for OCD. Given that approximately a quarter of patients with OCD receive little benefit from the currently available forms of treatment,20-22 and that those who do benefit rarely recover completely, new approaches for treating this population are urgently needed.

Here we present a method for training people to control a brain area involved in contamination anxiety and for probing the relationship between contamination anxiety and brain connectivity patterns.

We present a method for training subjects to control activity in a region of their orbitofrontal cortex associated with contamination anxiety using ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Video Movement Analysis Using Smartphones (ViMAS): A Pilot Study

The use of smartphones in clinical practice is steadily increasing with the availability of low cost/freely available &#34;apps&#34; that could be used to assess human gait. The primary aim of this manuscript is to test the concurrent validity of kinematic measures recorded by a smartphone application in comparison to a 3D motion capture system in the sagittal plane. The secondary aim was to develop a protocol for clinicians on the set up of the smartphone camera for video movement analysis.
The sagittal plane knee angle was measured during heel strike and toe off events using the smart phone app and a 3D motion-capture system in 32 healthy subjects. Three trials were performed at near (2-m) and far (4-m) smartphone camera distances. The order of the distances was randomized. Regression analysis was performed to estimate the height of the camera based on either the subject&#39;s height or leg length.
Absolute measurement errors were least during toe off (3.12 &#177; 5.44 degrees) compared to heel strike (5.81 &#177; 5.26 degrees). There were significant (p &#60; 0.05) but moderate agreements between the application and 3D motion capture measures of knee angles. There were also no significant (p &#62; 0.05) differences between the absolute measurement errors between the two camera positions. The measurement errors averaged between 3 - 5 degrees during toe off and heel strike events of the gait cycle.
The use of smartphone apps can be a useful tool in the clinic for performing gait or human movement analysis. Further studies are needed to establish the accuracy in measuring movements of the upper extremity and trunk.

Video Movement Analysis Using Smartphones ViMAS: A Pilot Study

This manuscript describes the method to test the concurrent validity of kinematic measures recorded by the smartphone application in comparison to a 3D motion capture system in the sagittal plane. This protocol will enable clinicians to set up smartphones for video capture of human movement.

The use of smartphones in clinical practice is steadily increasing with the availability of low cost/freely available &#34;apps&#34; that could be used to ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Methods Article

Movement Retraining using Real-time Feedback of Performance