نحن نعترف بامتنان الأجهزة الجزيئية التقنيات التحليلية / النعش على تبرعه السخي من الكواشف المستخدمة في الدراسة. ونحن نشكر أيضا الموارد الأنسجة مركز هارفارد لتوفير أنسجة الدماغ البشري بعد الوفاة التمويل : RO1MH76060 وP50MH080272.

<ol>
	<li>Imbeaud, S., Graudens, E., Boulanger, V., Barlet, X., Zaborski, P., Eveno, E., Mueleer, O., Schroeder, A., Auffray, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+of+RNA+quality+assessment+using+user-independent+classifiers+of+microcapillary+electrophoresis+traces.">Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.</a> Nucleic Acids Res. 33, e56-e56 (2005).</li></ol>

وقد رعت إنتاج هذه المادة عن طريق الفيديو بواسطة تقنيات الحياة.

هنا ، ونحن نحاول تخصيص بروتوكول لاستخراج الخلايا متجانسة من السكان غير متجانسة نسيج المخ بعد الوفاة باستخدام نظام XT النعش ومجموعات ذات صلة استخراج الحمض النووي الريبي. على النحو المبين أعلاه ، أحرزنا بعض التعديلات على البروتوكولات الأصلية التي تقدمها الشركة ، من أجل تحقيق أقصى قدر من الحمض النووي الريبي النزاهة. واسر واحد من الخلايا الناجح يعتمد فقط على تحقيق الاستفادة المثلى من الخطوات السابقة لالتقاط من أجل تحقيق أقصى قدر من خلايا الحمض النووي الريبي جيدة. هذه الخطوات تشمل مختلف المعايير المرتبطة الأنسجة sectioning وتلطيخ. باختصار ، فيما يتعلق بإعداد هذه الأنسجة تشمل ما يلي : 1) خفض درجة الحرارة عند التدرج sectioning ، 2) وضع المقاطع فقط 2 لكل شريحة ، 3) القيام بجميع الخطوات الجفاف على الجليد ، 4) واضاف المانع ريبونوكلياز إلى 5 ، وصمة عار) زيادة مدة الجفاف في الإيثانول النهائي 100 ٪ لمدة 3 دقائق. فيما يتعلق اسر ، فإننا نرى أن الجمع بين CapSure HS القبعات ، مع البرنامج على وضع ماكرو ، وينبغي ضمان تحقيق أفضل النتائج. بيئة الرطوبة التي تسيطر عليها أمر حاسم لالتقاط الخلايا واحد ، ويقلل من ارتفاع نسبة الرطوبة بشكل كبير الأنسجة &quot;رفع&quot;. درجات الحرارة المرتفعة يمكن أيضا أن يكون لها تأثير سلبي على نوعية الحمض النووي الريبي. خلال بروتوكول عزل الحمض النووي الريبي مع عدة PicoPure ، هناك خطوات غسل اثنين. فمن المهم للتأكد من أن تتم إزالة كافة العازلة يغسل قبل ان ينتقل الى آخر لأنبوب microcentrifuge شطف الحمض النووي الريبي ، لأن أي وجود المخزن في عينتك النهائي ستؤدي في أقل RNA انطلاق لالتضخيم. لضمان ذلك ، ونحن الطرد المركزي الخطوة النهائية لغسل 2.5 دقائق بدلا من دقيقة 2 على النحو المقترح في البروتوكول المقدمة. بالنسبة للجزء الاكبر ، وكان البروتوكول التضخيم خطي وفقا لتعليمات الشركة الصانعة. ومع ذلك ، ينبغي أن تؤخذ نقطتين حاسمة في الاعتبار عند استخدام هذا البروتوكول : 1) في محاولة للحد من الخسائر عن طريق الحمض النووي الريبي نقل الفائض من بين عينات أنابيب ، وذلك فقط باستخدام أنابيب 0.5ml قدمت بالاشتراك مع كتلة 0.5ml في cycler الحرارية و 2) عند نقل العينة إلى العمود تنقية لتنقية [كدنا] ، ومن المؤكد ان تدور باستمرار أنابيب العينات بعد نقل الأولى. وهذا سيؤدي في 2ul - 1 إضافية من العينة إضافة إلى العمود تنقية ، وبالتالي يمكن أن تزيد من الانتعاش [كدنا]. عند الاستفادة من هذه المجموعة في نهاية الوسم TURBO البيوتين لوضع العلامات من العينات الخاصة بك آرنا محدودة ، فإننا نقترح إضافة ميكرولتر اضافية قليلة من الماء قبل خطوة الطرد المركزي ، لزيادة الغلة استخراج الحمض النووي الريبي من العمود المسمى. سيكون في الدقيقة إضافية من الطرد المركزي يساعد أيضا في هذا الصدد ، خصوصا إذا كنت تعمل مع الأنسجة المتدهورة ، مثل الأنسجة FFPE. ونحن على ثقة من أن هذا البروتوكول سوف تمكن المستخدم لعزل هياكل صغيرة متجانسة ، مثل السكان خلية واحدة ، غير متجانسة داخل أنسجة الدماغ بعد الوفاة. هذا يقلل من آثار التخفيف من الهياكل المحيطة بها وغيرها من أنواع الخلايا ، الموجود في طبقات مختلفة داخل المخ ، مما يؤدي إلى النتائج المستهدفة نحو أنواع معينة من الخلايا قيد التحقيق. كما نوعية RNA الناتجة جيدة بما يكفي للدراسات ميكروأري ، يمكننا أن نبدأ في تحديد التوقيعات الجزيئية المحددة للسكان المتضررين في خلية معينة الحالات المرضية.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>4.5&#x201D; Forceps</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82027-392</td>
<td>&nbsp;</td>
<tr>
<td>Arcturus XT&trade; Laser-Capture Microdissection (LCM) System</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>13821-00</td>
<td>&nbsp;</td>
<tr>
<td>CapSure&trade; HS LCM Caps</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>LCM 0214</td>
<td>&nbsp;</td>
<tr>
<td>CapSure&reg; Macro LCM Caps</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>LCM 0211</td>
<td>&nbsp;</td>
<tr>
<td>Experion&trade; Automated Electrophoresis Station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7001</td>
<td>&nbsp;</td>
<tr>
<td>Experion&trade; RNA HighSens Chips</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7155</td>
<td>10 chips</td>
</tr>
<tr>
<td>Experion&#x2122; RNA HighSens Reagents</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7156</td>
<td>10 chips</td>
</tr>
<tr>
<td>Experion&#x2122; RNA StdSens Chips</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7153</td>
<td>10 chips</td>
</tr>
<tr>
<td>Experion&trade; RNA StdSens Reagents</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>700-7154</td>
<td>10 chips</td>
</tr>
<tr>
<td>Falcon&reg; polypropylene Conical tube</td>
<td>&nbsp;</td>
<td>Becton-Dickinson Labware</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>GeneAmp&reg; thin-walled reaction tube with domed cap</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>N8010611</td>
<td>&nbsp;</td>
<tr>
<td>GeneChip&reg; Human X3P array</td>
<td>&nbsp;</td>
<td>Affymetrix</td>
<td>900516</td>
<td>6 chips</td>
</tr>
<tr>
<td>Histogene&reg; LCM Frozen Section Staining kit</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>KIT0401</td>
<td>For staining solution, jars, ethanols and Xylene</td>
</tr>
<tr>
<td>Histogene&trade; LCM slides</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>12231-00</td>
<td>&nbsp;</td>
<tr>
<td>Micro Slide Box</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48444-004</td>
<td>&nbsp;</td>
<tr>
<td>Microm HM 505E cryostat</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22-050-350</td>
<td>Catalogue number for similar product as current product no longer available</td>
</tr>
<tr>
<td>Microprocessor Controlled 280 series Water bath</td>
<td>&nbsp;</td>
<td>Thermo Electron Corporation</td>
<td>51221048</td>
<td>Model 282</td>
</tr>
<tr>
<td>Molecular Sieve</td>
<td>&nbsp;</td>
<td>EMD&trade;</td>
<td>MX1583L-1</td>
<td>&nbsp;</td>
<tr>
<td>NanoDrop 1000 Spectrophotometer</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>n/a</td>
<td>The NanoDrop 1000 is not available anymore, but the NanoDrop 2000 is comparable.</td>
</tr>
<tr>
<td>Nuclease-free water</td>
<td>&nbsp;</td>
<td>Ambion&reg;</td>
<td>AM9932</td>
<td>&nbsp;</td>
<tr>
<td>PEN Membrane glass slides</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>LCM 0522</td>
<td>&nbsp;</td>
<tr>
<td>PicoPure&reg; RNA Isolation kit</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>KIT0204</td>
<td>&nbsp;</td>
<tr>
<td>RiboAmp&reg;HSPLUS with Biotin labeling</td>
<td>&nbsp;</td>
<td>MDS Analytical Technologies</td>
<td>KIT0511B</td>
<td>12 reactions Includes TURBO biotin labeling&trade; kit</td>
</tr>
<tr>
<td>RNase Zap&reg;</td>
<td>&nbsp;</td>
<td>Ambion&reg;</td>
<td>AM9780/2</td>
<td>&nbsp;</td>
<tr>
<td>RNase-Free DNase Set</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>79254</td>
<td>&nbsp;</td>
<tr>
<td>RNasin&reg; Plus</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>N261B</td>
<td>&nbsp;</td>
<tr>
<td>SuperScript&trade; III Reverse Transcriptase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18080-044</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

obtaining high quality rna from single cell populations in human postmortem brain tissue

اقترحنا للتحقيق في الحد من المادة الرمادية في التلفيف الصدغي العلوي ينظر في مرضى الفصام ، من خلال استجواب ملامح التعبير الجيني للخلايا العصبية الهرمية في الطبقة الثالثة. ومن المعروف أن قشرة الدماغ هي بنية متجانسة بشكل استثنائي يضم مختلف المناطق والطبقات وأنواع الخلايا ، كل منها يتميز متميزة التراكيب الخلوية والجزيئية وبالتالي الفرق ملامح التعبير الجيني. للتحايل على آثار الخلط من التجانس الأنسجة ، واستخدمنا الليزر التقاط microdissection (LCM) من أجل عزل لدينا خلية معينة من نوع الخلايا العصبية أي الهرمي. تم القبض على ما يقرب من 500 العصبونات الهرمية ملطخة Histogene حل التلوين باستخدام نظام أركتوروس LCM XT. ثم تم عزل الحمض النووي الريبي من الخلايا والقبض على خضع جولتين من T7 المستندة إلى تضخيم الخطية باستخدام النعش / الجزيئية أجهزة مجموعات. أشار LabChip Experion (بيو راد) هلام ومخطط رحلاني نوعية جيدة الحمض النووي الريبي (م) ، ويبلغ طوله نص تمديد 600nt الماضية اللازمة لميكروأرس. وبلغ متوسط ​​كمية مرنا الحصول 51μg ، مع نقاء مقبول متوسط ​​العينة على النحو المبين في النسبة A260/280 ، من 2.5. وعرضت لمحة الجينات البشرية باستخدام مسبار GeneChip X3P مجموعة من Affymetrix.

Watch this Scientific Journal Video about Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue at JoVE.com

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

نحن تصف العملية باستخدام الليزر التقاط microdissection لعزل واستخلاص الحمض النووي الريبي من السكان خلية واحدة متجانسة ، العصبونات الهرمية ، في الثالث من طبقة التلفيف الصدغي العلوي في العقول بعد الوفاة الإنسان. نحن في وقت لاحق تضخيم خطي (T7 على أساس) مرنا ، ويهجن العينة إلى ميكروأري Affymetrix X3P الإنسان.

الحصول على جودة عالية من الحمض النووي الريبي بالسكان خلية واحدة في الإنسان تشريح أنسجة المخ

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

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Research

JoVE Journal

Biology

30.5K Views.  McLean Hospital. نحن تصف العملية باستخدام الليزر التقاط microdissection لعزل واستخلاص الحمض النووي الريبي من السكان خلية واحدة متجانسة ، العصبونات الهرمية ، في الثالث من طبقة التلفيف الصدغي العلوي في العقول بعد الوفاة الإنسان. نحن في وقت لاحق تضخيم خطي (T7 على أساس) مرنا ، ويهجن العينة إ...

Video: Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

الحصول على البيض من الإناث المورق القيطم

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

عزل النواة من الخلايا العصبية البشرية تشريح أنسجة المخ

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

جيل وحيدة الخلية معلقات من الأنسجة العصبية ماوس

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

تسجيلات شفط وحيدة الخلية المستقبلة من المخروط ماوس

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

قوية من الجيل الكبدية مثل خلايا الجنينية البشرية من السكان الخلايا الجذعية

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

جيل من جودة عالية لونين القالب الحمض النووي مناعي لتسلسل عالية الإنتاجية (رقاقة وما يليها)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

بروتوكول إزالة الميكروسيرس من الليزر الذي ينتج RNA عالي الجودة من عظام الماوس الطازجة المجمدة

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

سير العمل البروتيومي الكمي باستخدام رصد التفاعل المتعدد القائم على الكشف عن البروتينات من أنسجة الدماغ البشري

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

جمع الأنسجة واستخراج الحمض النووي الريبي من مفصل الركبة هشاشة العظام البشرية

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.