<ol>
	<li>Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 1827-1831 (1992).</li><li>Clark, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic. Acids. Res. 22, 2990-2997 (1994).</li><li>Grunau, C., Clark, S. J., Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisulfite+genomic+sequencing:+systematic+investigation+of+critical+experimental+parameters.">Bisulfite genomic sequencing: systematic investigation of critical experimental parameters.</a> Nucleic. Acids. Res. 29, E65-E65 (2001).</li><li>Clark, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+bisulphite+modification+and+analysis.">DNA methylation: bisulphite modification and analysis.</a> Nat. Protoc. 1, 2353-2364 (2006).</li></ol>

الإعلان عن أي تضارب في المصالح.

تحليل الحمض النووي مثلأيشن بواسطة التسلسل الجيني للحمض النووي bisulphite تحويل طريقة راسخة ومرنة تسهل تحديد وتقدير من الحامض النووي في قرار النوكليوتيدات واحد. ومع ذلك ، وتحويل bisulphite وتحليل لاحقة تعتمد على فرضية أنه قد تم تحويل الحمض النووي بشكل كامل مع كل السيتوزين unmethylated يجري deaminated إلى اليوراسيل وcytosines فقط ميثليته المتبقية من الامم المتحدة وعلى رد الفعل. إذا كان التحويل غير كاملة ، يمكن تنشأ مشاكل مع التحليل ، إذ يمكن تحويله cytosines unmethylated تفسير غير صحيح كما cytosines ميثليته. يمكن أن يكون الأمثل Bisulphite التحويل في مراحل مختلفة لتحقيق أقصى قدر نسبة التحويل. أن الحمض النووي لتحويلها بالكامل يجب أن يكون أول واحد الذين تقطعت بهم السبل بحيث يتعرضون للمخلفات السيتوزين bisulphite الأيونات. الخطوة الأولى ، تمسخ الحمض النووي ، أمر بالغ الأهمية ، ويمكن أن تكون مصدرا للتحويل الحمض النووي ناقصا إذا لم يتم التشويه والتحريف بشكل كامل. لضمانأن الحمض النووي هو التشويه والتحريف بشكل كامل من المهم أن رد فعل المعلمات بما في ذلك إزالة جميع البروتينات المرتبطة بها ، حسب تركيز الملح ، ودرجة حرارة الحضانة والوقت المناسب للحفاظ على الحمض النووي في التشكل الذين تقطعت بهم السبل واحدة. من المهم استخدام الطازجة 3M هيدروكسيد الصوديوم والسماح الوقت الكافي للحضانة. إذا كان الحمض النووي تقاوم تمسخ ، قد يكون بعض التعديلات على البروتوكول بما في ذلك تمديد فترة حضانة لمدة 30 دقيقة أو تفتيت الحمض النووي قبل تمسخ تسهيل تمسخ الحمض النووي. بالإضافة إلى ذلك ، قد تقطعت بهم السبل الحمض النووي واحد (ssDNA) إعادة يصلب لمضاعفة الحمض النووي الذين تقطعت بهم السبل (dsDNA) خلال رد فعل التحويل bisulphite. ويمكن تنفيذ رد فعل على ارتفاع درجة الحرارة (90-95 درجة مئوية) إما بصورة دورية أو مستمرة خلال تفاعل bisulphite المساعدات صون ssDNA. ومع ذلك ، فإنه من المهم أن ندرك أن يتم تسريعها تدهور الحمض النووي في ارتفاع درجات الحرارة هذه. ويمكن أيضا الكواشف المختلفة يمكن ان تضاف الى عرقلة إعادة الصلب من الجدائل الحمض النووي ه ، مثل اليوريا. يمكن للتركيز الحمض النووي ونوعيته يؤثر أيضا على كفاءة التفاعل bisulphite والعائد النهائي PCR. تدهور الحمض النووي هو وجود قيود على تحويل بروتوكول bisulphite. العلاج الكيميائي للفواصل حبلا الحمض النووي يدخل في مختلف ssDNA وبعض الظروف القاسية اللازمة لتحويل bisulphite كاملة بما في ذلك تركيز عالية bisulphite مرات حضانة طويلة يمكن أن يسرع تدهور الحمض النووي. وفقا للشروط القياسية وصف رد الفعل ، وتدهور الحمض النووي ليست مشكلة شائعة ولكن ، إذا أدخلت تعديلات البروتوكول ، مثل متواليات التي يتم صهر للتحويل ، لقوالب الحمض النووي التي تدهورت أو من نوعية رديئة مثل عينات FFPE ، ثم تدهور الحمض النووي يمكن أن يصبح وجود قيود كبيرة. يمكن الفورمالين الثابتة ، جزءا لا يتجزأ من البارافين (FFPE) وعينات من الحمض النووي المتدهورة على نحو فعال وتضخيم bisulphite تحويلها باستخدام هذا البروتوكولولكن ينصح التعديلات للتأكد من أن الحمض النووي لا مزيد من التدهور. ينصح استخدام الحمض النووي الريبي الناقل مثل الحمض النووي الريبي ر. ويمكن أيضا أن يضاف الجليكوجين باعتبارها الناقل عند تركيز منخفض الحمض النووي (&lt;200 نانوغرام). لأن الحمض النووي من الأنسجة المعزولة FFPE بالفعل مجزأة ومزيد من الانقسام يحدث أثناء نزع الأمين ، يجب الحرص على الحد من مزيد من التدهور. لا مزيد من الحمض النووي قبل أي جزء تمسخ والحد من فترة حضانة للتفاعل bisulphite إلى 4 ساعات. تصميم amplicons لم أكبر من 300 سنة مضت بسبب الحمض النووي مجزأة. عامل آخر يمكن أن تؤثر على تضخيم PCR أن التحويل من bisulphite cytosines unmethylated يقلل من تعقيد قالب الحمض النووي. ويمكن تصميم التمهيدي بعد بروتوكول المبينة في القسم 4.1 ، زيادة طول التمهيدي وPCR المتداخلة مساعدة جميع لزيادة الغلة والنوعية PCR. أخيرا ، منذ القالب الحمض النووي هو تغيير خلال bisulphويمكن تحويل الفنار ، والتحيز التضخيم تكون مصدرا للقلق. ضمان أن يتم فحص PCR التضخيم النسبي من القوالب وميثليته unmethylated مع مزيج 50/50 سيطرة الحمض النووي M / U ، كما ورد في البروتوكول ، انظر الشكل 3. تأكد أيضا أن التضخيم من القوالب تحويلها فقط يحدث باستخدام انزيم HpaIII التقييد والكهربائي للهلام (الشكل 3). قد تحتاج إلى ظروف PCR يكون الأمثل من خلال تغيير درجة الحرارة وتركيز / أو المغنيسيوم أو إطالة وقت التمديد. بعض القوالب ويبدو أن مشكلة خاصة وموقف التمهيدي قد تحتاج إلى أن تعدل أو تتحول الاشعال قد تحتاج لاستخدامه. تقنيات الجيل القادم التسلسل تتطور بسرعة لتحقيق تسوية مماثلة لتسلسل الجينوم bisulphite ، وثالث التسلسل جيل جزيء واحد لديه القدرة على السماح لتحليل الحمض النووي لكل من الابتدائي ومثيلة من دون الحاجة إلى تسلسل bisulphite. ومع ذلك ، وتوافر واسع النطاق والمواصفاتificity من هذه التقنيات لا تزال بعض الوقت بعيدا. تسلسل الجينوم Bisulphite هو معيار الذهب في التحليل ومثلأيشن هو بروتوكول قوية. وقد أحرز تقدم في طريقة إلى النقطة التي تم فيها حل المشاكل المشتركة والقيود والطلبات قد وسعت من التحليلات الجينية الفردية إلى إنتاجية عالية وتحليل كامل الجينوم التي وصفها كلارك وآخرون 4 2006. وترد المبادئ التوجيهية والتوصيات لحل المشاكل إضافية في الجدول رقم 1. يقترح أن النهج الأمثل هو اتباع البروتوكول القياسي في البداية فقط وإدخال التعديل إذا وعندما تنشأ مشكلة. <img src="/files/ftp_upload/3170/3170table2.jpg" alt="الجدول 2" /> الجدول 1. المبادئ التوجيهية استكشاف

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Metabisulphite الصوديوم (اياكس Finechem) </li><li style=";text-align:right;direction:rtl"> هيدروكينون (ميرك) </li><li style=";text-align:right;direction:rtl"> الحمض النووي المعالج تنظيف النظام (Promega) </li></ol>

dna methylation: bisulphite modification and analysis

علم التخلق يصف التغيرات في وظيفة الجينات الموروثة التي تحدث بشكل مستقل لتسلسل الحمض النووي. الأساس الجزيئي للتنظيم الجينات جينية معقدة ، ولكن ينطوي أساسا تعديلات على الحمض النووي أو البروتينات نفسها التي الحمض النووي الزميلة. التعديل اللاجينية الغالبة من الحمض النووي في جينومات الثدييات هي مثيلة من النيوكليوتيدات السيتوزين (5 - MEC). الحامض النووي يوفر التعليم لآلية التعبير الجيني إلى أين ومتى ينبغي التعبير عن الجين. والهدف الرئيسي لتسلسل الحامض النووي في الثدييات هو 5&#39; - CPG - 3 &#39;dinucleotides (الشكل 1). لا dinucleotides CPG موزعة بشكل متجانس في جميع أنحاء الجينوم ، ولكنها تتركز في مناطق تسلسل الجينوم المتكررة وCPG &quot;الجزر&quot; التي ترتبط عادة مع المروجين الجين (الشكل 1). وتنشأ أنماط الحامض النووي في وقت مبكر في عملية التنمية ، التضمين أثناء تمايز أنسجة محددة وتعطلت في دول كثيرة من بينها مرض السرطان. إلى شمطلوبة nderstand الدور البيولوجي في الحامض النووي ودورها في المرض ، والإنسان الدقيق وفعالة وقابلة للتكرار الأساليب لكشف وتحديد الفرد 5 MeCs. هذا البروتوكول لتحويل bisulphite هو &quot;المعيار الذهبي&quot; لتحليل الحامض النووي ويسهل تحديد وتقدير من الحامض النووي في قرار النوكليوتيدات واحد. كيمياء نزع الأمين السيتوزين bisulphite الصوديوم التي تتضمن ثلاث خطوات (الشكل 2). (1) Sulphonation : إضافة إلى السندات bisulphite 5-6 المزدوج لنزع الأمين (2) السيتوزين الهيدروليكية : نزع الأمين حلمهي المشتقة السيتوزين ، مما أدى إلى إعطاء bisulphite مشتق اليوراسيل - bisulphite (3) Desulphonation قلوي : إزالة للسلفونات المجموعة علاج القلوية ، لإعطاء اليوراسيل. Bisulphite deaminates تفضيلي السيتوزين إلى اليوراسيل في الحمض النووي الذين تقطعت بهم السبل واحد ، في حين MEC - 5 ، هو صهر لbisulphite بوساطة نزع الأمين. على تضخيم PCR ، يتم تضخيمه اليوراسيلكما الثايمين بينما 5 - MEC مخلفات تبقى كما cytosines ، مما يسمح حاليا CpGs ميثليته من CpGs unmethylated بواسطة جود &quot;جيم&quot; السيتوزين مقابل بقايا الثايمين &quot;T&quot; خلال التسلسل. تعديل الحمض النووي عن طريق تحويل bisulphite هو بروتوكول الراسخة التي يمكن استغلالها لطرق كثيرة للتحليل الحامض النووي. وقد تجلى منذ أول اكتشاف MEC - 5 عن طريق التحويل بواسطة bisulphite فرومر وآخرون (1) وكلارك وآخرون. 2 ، وطرق التحويل القائمة حول bisulphite حساب الحمض النووي الجيني لغالبية بيانات جديدة عن الحامض النووي. يمكن استخدام أساليب مختلفة للتحليل PCR آخر ، اعتمادا على درجة من الدقة وحلها مثلأيشن المطلوبة. الاستنساخ والتسلسل لا يزال الأسلوب الأكثر توافرا التي يمكن أن تعطي واحدة لقرار النوكليوتيدات مثيلة عبر جزيء الحمض النووي.

Watch this Scientific Journal Video about DNA Methylation: Bisulphite Modification and Analysis at JoVE.com

DNA Methylation: Bisulphite Modification and Analysis

المعيار الذهبي لتحليل الحامض النووي هو التسلسل الجيني للbisulphite الحمض النووي التي تم تحويلها. هذا الأسلوب يستفيد من زيادة الحساسية للمقارنة مع السيتوزين methylcytosine - 5 (5 - MEC) إلى نزع الأمين bisulphite تحت الظروف الحمضية. ويمكن تمييز cytosines Unmethylated من cytosines ميثليته بعد التضخيم PCR هدف الحمض النووي الجيني.

الحامض النووي : Bisulphite تعديل وتحليل

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

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103.9K Views.  Garvan Institute of Medical Research. المعيار الذهبي لتحليل الحامض النووي هو التسلسل الجيني للbisulphite الحمض النووي التي تم تحويلها. هذا الأسلوب يستفيد من زيادة الحساسية للمقارنة مع السيتوزين methylcytosine - 5 (5 - MEC) إلى نزع الأمين bisulphite تحت الظروف الحمضية. ويمكن تمييز cytosines Unmethylated من cytosines ميثليته بعد التضخي?...

Video: DNA Methylation: Bisulphite Modification and Analysis

PDMS Device Fabrication and Surface Modification

PDMS تصنيع الأجهزة وتعديل السطح

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

والخلايا العصبية ونجمية المشارك الثقافة الفحص لتحليل محتوى عال من العصبية

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

تحليل مزدوج الجديلة إصلاح الحمض النووي (DSB) فاصل في خلايا الثدييات

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

تقرير من الحامض النووي من الجينات في مطبوع Arabidopsis السويداء

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

تعديل وإعادة التركيب الوراثي للاللعابية الثقافات الجهاز الغدة

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

وسيلة فعالة للالكمي، التحليل خلية واحدة من لونين تعديل والعمارة النووية في جبل لالجامع بويضات في<em&gt; نبات الأرابيدوبسيس</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

التصوير في الوقت الحقيقي من واحدة المهندسة رنا النصوص في الخلايا الحية عن طريق Ratiometric ثنائي الجزيء منارات

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

انخفاض الجاذبية المظاهرات الأجهزة البيئة في نموذج المنمنمة تدفق عداد الكريات ورفيق ميكروفلويديك تقنية خلط

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

في المختبر مثلأيشن الفحص لدراسة البروتين أرجينين مثلأيشن

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

المحسن انخفاض التمثيل بيسلفيت تسلسل لتقييم الحامض النووي في قرار قاعدة زوج

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...