نشكر الدكتور أغنيس فيالي ومختبر علم الجينوم MSKCC الأساسية للحصول على المساعدة الفنية. وقد تم تطوير هذا البروتوكول بدعم من مركز أبحاث السرطان Beene جيفري ومؤسسة مزارع العائلة.

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وقد تلقى الدكتور بيرغر رسوم الاستشارات وتمويل البحوث من مؤسسة الطب، وهي شركة تشخيص السرطان.

لدينا IMPACT فحص تنتج نسبة عالية المحاذاة، وارتفاع معدل على الهدف، التغطية المستهدفة عالية، وحساسية عالية للكشف عن الطفرات، indels، ونسخ التعديلات العدد. لقد أثبتنا قدرة لدينا فحص الحمض النووي لIMPACT تسلسل من كلا الطازجة المجمدة وأرشفة عينات الحمض النووي FFPE من المدخلات من?...

تحديد &quot;سائق&quot; الأحداث الجينية في الورم المسرطنة الرئيسية وورم القامع الجينات تلعب دورا أساسيا في تشخيص وعلاج العديد من أنواع السرطان 1. وقد مكنت الجهود البحثية على نطاق واسع باستخدام المتوازية &quot;الجيل القادم&quot; التسلسل تحديد الكثير من هذه الجينات المرتبطة بالسرطان في السنوات الأخيرة 2. ومع ذلك، تتطلب هذه المنصات التسلسل عادة كميات كبيرة من الحمض النووي المعزولة من أنسجة الطازجة المجمدة، مما يشكل عائقا كبيرا في تشخيص وتحليل الطفرات الحمض النووي من الأنسجة الحفاظ عليها، مثل البارافين الثابتة الفورمالين جزءا لا يتجزأ من (FFPE) عينات الورم. سوف تحسين الجهود لبكفاءة وبشكل موثوق تميز المعلومات الجينية &quot;للتنفيذ&quot; من عينات الورم FFPE تمكين تحليل بأثر رجعي من العينات المخزنة سابقا، ومواصلة تشجيع النهج الفردي لإدارة السرطان. تقليديا، د الجزيئيةوقد اعتمدت مختبرات iagnostic على والمنهجيات المنخفضة الإنتاجية تستغرق وقتا طويلا مثل سانجر تسلسل وPCR الوقت الحقيقي لتحديد ملامح طفرة الحمض النووي. وفي الآونة الأخيرة، تم تطوير أساليب إنتاجية أعلى باستخدام PCR المضاعفة أو الطيفي التنميط الجيني الشامل للتحقيق الطفرات الجسدية المتكررة في جينات السرطان الرئيسية 3-5. هذه النهج، ومع ذلك، وتقتصر في ذلك فقط المحددة مسبقا &quot;نقطة ساخنة&quot; الطفرات ويعاير، مما يجعلها غير صالحة للكشف عن الطفرات في الورم تعطيل الجينات القامع. تسلسل المتوازية يقدم العديد من المزايا أكثر من هذه الاستراتيجيات بما في ذلك القدرة على استجواب الإكسونات كامل للطفرات أمر شائع ونادر، والقدرة على الكشف عن الطبقات الإضافية من التعديلات الجيني مثل المكاسب والخسائر في عدد النسخ، وزيادة حساسية الكشف في عينات غير متجانسة 6، 7 . يمثل تسلسل الجينوم كله النهج الأكثر شمولا لاكتشاف طفرة، على الرغم من أنه أمر نسبيويتحمل تكلفة لاي مطالب الحسابية الكبيرة لتحليل البيانات والتخزين. بالنسبة إلى التطبيقات السريرية، حيث لا يوجد سوى جزء صغير من الجينوم قد تكون ذات فائدة السريرية، كانت اثنين من الابتكارات في مجال التكنولوجيا خاصة التسلسل التحويلية. أولا، من خلال التهجين القائم على التقاط اكسون، يمكن للمرء أن عزل الحمض النووي الموافق الجينات المرتبطة بالسرطان الرئيسية للطفرة التنميط استهدافا 8. ثانيا، من خلال ربط الباركود الجزيئية (أي تسلسل الحمض النووي 6-8 النيوكليوتيدات في الطول)، يمكن للمرء أن تجمع مئات العينات في المدى التسلسل واستفادة الكاملة من قدرة متزايدة من الصكوك تسلسل المتوازية 10. عند الجمع، وهذه الابتكارات تمكين الأورام ليكون موجز لانخفاض تكلفة وأعلى إنتاجية في، مع المتطلبات الحسابية أصغر 11. علاوة على ذلك، من خلال إعادة توزيع التغطية تسلسل فقط تلك الجينات الأكثر أهمية لتطبيق معين، يمكن للمرء achieلقد بمزيد من التعمق تسلسل لأعلى حساسية الكشف عن انخفاض وتيرة الأحداث أليل. نحن هنا وصفا لدينا IMPACT مقايسة (الطفرة التنميط المتكاملة من الأهداف المنحى السرطان)، الذي يستخدم القبض على اكسون حمامات مكتبة تسلسل barcoded بواسطة التهجين باستخدام [أليغنوكليوتيد مخصصة لالتقاط كل الإكسونات الترميز البروتين وحدد الإنترونات من 279 الجينات المرتبطة بالسرطان الرئيسية (الجدول 1 ). هذه الاستراتيجية تمكن تحديد الطفرات، indels، والتعديلات في عدد النسخ، وحدد إعادة ترتيب الهيكلية التي تنطوي على هذه الجينات 279. أسلوبنا هو متوافق مع الحمض النووي المعزولة من أنسجة سواء الطازجة المجمدة وFFPE وكذلك حرف هاء إبرة دقيقة وعينات الخلايا الأخرى.

<table border="0" cellpadding="0" cellspacing="0" width="812">
	<tbody> 
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEBNext End Repair Module</td>
			<td style="width:247px;">New England Biolabs</td>
			<td style="width:137px;">E6050L</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEBNext dA-Tailing Module</td>
			<td style="width:247px;">New England Biolabs</td>
			<td style="width:137px;">E6053L</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEBNext Quick Ligation Module</td>
			<td style="width:247px;">New England Biolabs</td>
			<td style="width:137px;">E6056L</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Agencourt AMPure XP</td>
			<td style="width:247px;">Beckman Coulter Genomics</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEXTflex PCR-Free Barcodes &#8211; 24</td>
			<td style="width:247px;">Bioo Scientific</td>
			<td style="width:137px;">514103</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HiFi Library Amplification Kit</td>
			<td style="width:247px;">KAPA Biosystems</td>
			<td style="width:137px;">KK2612</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">COT Human DNA, Fluorometric Grade</td>
			<td style="width:247px;">Roche Diagnostics</td>
			<td style="width:137px;">05 480 647 001</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NimbleGen SeqCap EZ Hybridization and Wash kit</td>
			<td style="width:247px;">Roche NimbleGen</td>
			<td style="width:137px;">05 634 261 001</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">SeqCap EZ Library Baits</td>
			<td style="width:247px;">Roche NimbleGen</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">QIAquick PCR Purification Kit</td>
			<td style="width:247px;">Qiagen</td>
			<td style="width:137px;">28104</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Qubit dsDNA Broad Range (BR) Assay Kit</td>
			<td style="width:247px;">Life Technologies</td>
			<td style="width:137px;">Q32850</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Qubit dsDNA High Sensitivity (HS) Assay Kit</td>
			<td style="width:247px;">Life Technologies</td>
			<td style="width:137px;">Q32851</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Agilent DNA HS Kit</td>
			<td style="width:247px;">Agilent Technologies</td>
			<td style="width:137px;">5067-4626, 4627</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Agilent 2100 Bioanalyzer</td>
			<td style="width:247px;">Agilent Technologies</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Covaris E220</td>
			<td style="width:247px;">Covaris</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Magnetic Stand-96</td>
			<td style="width:247px;">Ambion</td>
			<td style="width:137px;">AM10027</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Illumina Hi-Seq 2000</td>
			<td style="width:247px;">Illumina</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
	</tbody>
</table>

detecting somatic genetic alterations in tumor specimens by exon capture and massively parallel sequencing

وقد أثبتت الجهود للكشف والتحقيق الطفرات أنكجنيك مفتاح قيمة لتسهيل العلاج المناسب لمرضى السرطان. إنشاء الإنتاجية العالية، المتوازية &quot;الجيل القادم&quot; التسلسل ساعد على اكتشاف العديد من هذه الطفرات. لتعزيز المرافق السريرية ومتعدية لهذه التكنولوجيا، ويجب أن تكون منصات عالية الإنتاجية، وفعالة من حيث التكلفة، ومتوافقة مع البارافين الثابتة الفورمالين جزءا لا يتجزأ من (FFPE) عينات الأنسجة التي قد تسفر عن كميات صغيرة من الحمض النووي المتدهورة أو التالفة. هنا، نحن تصف إعداد مكتبات الحمض النووي barcoded والمضاعفة تليها التقاط القائم على التهجين من الإكسونات المستهدفة للكشف عن الطفرات المرتبطة بالسرطان في الأورام المجمدة والطازجة FFPE بواسطة تسلسل المتوازية. هذه الطريقة تمكن من تحديد الطفرات تسلسل، نسخ التعديلات العدد، وحدد إعادة ترتيب الهيكلية التي تنطوي على جميع الجينات المستهدفة. استهداف تسلسل اكسون يقدم روقال انه يستفيد من إنتاجية عالية وتكلفة منخفضة، وتغطية تسلسل عميق، وبالتالي منح حساسية عالية للكشف عن الطفرات التردد المنخفض.

تم القبض على واحد مجموعة من 24 مكتبات barcoded تسلسل (12 زوجا للورم عادي) باستخدام مجسات المقابلة لجميع الإكسونات الترميز البروتين من 279 جينات السرطان والتسلسل كما يقرأ 2 × 75 نقطة أساس على حارة واحدة من خلية تدفق HiSeq 2000. وكان يتم تجميع الورم والمكتبات العادية في نسبة 2:1. وأظهر?...

Watch this Scientific Journal Video about Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing at JoVE.com

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

وصفنا إعداد مكتبات الحمض النووي barcoded واللاحقة المستندة إلى التهجين التقاط اكسون للكشف عن الطفرات المرتبطة بالسرطان الرئيسية في عينات الورم السريرية التي المتوازية &quot;الجيل القادم&quot; التسلسل. استهداف تسلسل اكسون يقدم فوائد إنتاجية عالية وتكلفة منخفضة، وتغطية تسلسل عميق، وبالتالي العائد حساسية عالية للكشف عن الطفرات التردد المنخفض.

الكشف عن التعديلات الوراثية الجسدية في عينات الورم عن طريق التقاط اكسون وتسلسلها الموازي واسع

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

detecting-somatic-genetic-alterations-tumor-specimens-exon-capture

Research

JoVE Journal

Biology

19.3K Views.  Memorial Sloan-Kettering Cancer Center.  وصفنا إعداد مكتبات الحمض النووي barcoded واللاحقة المستندة إلى التهجين التقاط اكسون للكشف عن الطفرات المرتبطة بالسرطان الرئيسية في عينات الورم السريرية التي المتوازية "الجيل القادم" التسلسل. استهداف تسلسل اكسون يقدم فوائد إنتاجية عالي?...

Video: Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T - اللمفاويات التقاط باستخدام رقاقة ميكروفلويديك المتاح لفيروس نقص المناعة البشرية

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

الجامعة جبل مزدوجة في الموقع التهجين لافراخ الدجاج في وقت مبكر

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

كفاءة الجيل المستحثة الإنسان افرة القوة الخلايا الجذعية من الخلايا الجسدية البشرية مع سينداي الفيروسات

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

Linear Amplification Mediated PCR &amp;#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.

الخطي التضخيم وساطة PCR - توطين العناصر الجينية وتوصيف غير معروف المرافقة الحمض النووي

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

إعداد جبل مسطح لمراقبة وتحليل الأجنة الزرد عينات الملون بواسطة جبل الجامعة<em&gt; في الوضع الطبيعي</em&gt; التهجين

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

فحوصات مراسل المتوازية نطاق واسع في خلايا الثدييات مثقف

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

برازي تحليل جلايكورتيكود: رصد الغدة الكظرية غير الغازية في الخيليات

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

استهدفت RNA تسلسل الفحص لتحديد خصائص التعبير الجيني والجينوم التعديلات

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.