This work was funded by a National Institutes of Health R01 (DC009453 support to CWB).

ملاحظة: قبل تقديم عينات من اللعاب جميع المواد أعطى موافقة مستنيرة تتفق مع المبادئ التوجيهية للعلاج من المواضيع الإنسان في مستشفى الأطفال على الصعيد الوطني. 1. اللعاب جمع وتخزين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل جمع اللعاب، تأكد من فم هذا الموضوع هو حر من المواد الغذائية أو المواد الغريبة الأخرى من خلال وجود موضوع شطف الفم بالماء وتجنب الأكل أو الشرب لمدة 30 دقيقة قبل جمع العينة. </li><li style=";text-align:right;direction:rtl"> فتح 15 مل أنبوب الطرد المركزي مع 2.5 مل من الحمض النووي استقرار عازلة 2 مع التأكد من تجنب لمس داخل الغطاء أو أنبوب، ويكون موضوع يبصقون 2.5 مل من اللعاب في حل العازلة. ملاحظة: يمكن جمع أكثر من 2.5 مل من اللعاب يؤدي إلى تدهور عينة من نسبة كافية من عينة لعازلة الاستقرار DNA. وجمع اللعاب قليلا جدا لحد عوائد المتوقعة من البروتوكول. لتقييم حجم جمع واستخدام التدرجات مرقمة على جانب ركان الأنبوب. </li><li style=";text-align:right;direction:rtl"> استبدال الغطاء وتخلط بواسطة قلب حتى يتم الخليط المتجانس. اهتزاز قوي ليست ضرورية. تخزين العينات في RT للتخزين على المدى القصير أو 4 درجة مئوية لمدة التخزين على المدى الطويل (&gt; 3 أشهر). </li></ol> 2. الاستعدادات الأولية وتحلل الخلية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل بدء الاستخراج، تسخين حمام مائي إلى 37 درجة مئوية، وإعداد دلو الجليد. سوف تكون هناك حاجة إلى ثلاثة 15 مل أنابيب الطرد المركزي المخروطية لكل عينة المستخرجة. وسوف تستخدم هذه الأنابيب الثلاثة لعقد الخلية والبروتين بيليه، المباراة النهائية المستخرجة gDNA، والأيزوبروبانول والإيثانول supernatants. </li><li style=";text-align:right;direction:rtl"> استرجاع عينات من التخزين، وعكس عينات عدة مرات ثم دوامة على سرعة متوسطة لمدة 15 ثانية. </li><li style=";text-align:right;direction:rtl"> الاستغناء 2.5 مل من العينة إلى نظيفة 15 مل أنبوب الطرد المركزي، وإضافة 5 مل من خلية تحلل الحل. مزيج العينة 50 مرات انعكاس، واحتضان في RT لمدة 30 دقيقة. </li></ol> إزالة 3. RNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 40 ميكرولتر من ريبونوكلياز وسولution في 100 ملغ / مل، واحتضان عند 37 درجة مئوية لمدة 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة عينة من 37 درجة مئوية الماء وحمام بارد على الجليد لمدة 3 دقائق. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد ريبونوكلياز والحضانة، وزيادة درجة حرارة حمام الماء إلى 65 درجة مئوية للخطوة الإماهة الحمض النووي للبروتوكول. </li></ol></li></ol> 4. البروتين وإزالة الدهون <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر من بروتين K الحل عند 20 ملغ / مل، مزيج عدة مرات من قبل انعكاس، واحتضان في RT مدة لا تقل عن 30 دقيقة. ملاحظة: هذه هي نقطة التوقف المحتملة للبروتوكول. بعد إضافة من الحل K بروتين، ويمكن تخزين العينة في 4 درجات مئوية حتى يمكن الانتهاء من الاستخراج. لم تظهر تخزين في 4 درجات مئوية لمدة تصل إلى 24 ساعة أن يكون لها تأثير كبير على عائدات استخراج أو نوعية الحمض النووي. لم يتم تقييم التخزين على المدى الطويل في هذه المرحلة. </li><li style=";text-align:right;direction:rtl"> إضافة 1.7 مل من الهطول البروتين الحل، دوامة بقوة لمدة 20 ثانية بسرعة عالية، ومكان على الجليد لمدة 10 دقيقة. </lط&gt; <li style=";text-align:right;direction:rtl"> بمجرد أن يبرد العينات على الجليد لمدة 10 دقيقة، الطرد المركزي لمدة 10 دقيقة في 3،000 x ج و 4 درجات مئوية. يجب على البروتينات عجلت تشكيل بيليه ضيق للمتابعة. إذا كان بيليه ليست ضيقة أو الحل لا تزال غائمة، العينات يمكن تبريده على الجليد لمدة 5 دقائق أكثر وتكرار الطرد المركزي. يجب أن تبقى العينات على الجليد لضمان بيليه مشددة. </li></ol> 5. عزل وتنقية gDNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في نظيفة 15 مل أنبوب الطرد المركزي، الماصة 5 مل من كحولالأيسوبروبيل و 8 ميكرولتر من الحل النقي الجليكوجين في 20 ملغ / مل. </li><li style=";text-align:right;direction:rtl"> صب طاف تحتوي على gDNA من الخطوة 4.3 في أنبوب يحتوي على الأيزوبروبانول والجليكوجين الحل، مخلفين وراءهم البروتين عجلت بيليه. مرة واحدة تمت إضافة طاف، مزيج بلطف العينة 50 مرات من قبل انعكاس وأجهزة الطرد المركزي لمدة 30 دقيقة في 3،000 x ج و 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> صب طاف ببطء في نظيفة أنبوب 15 مل. بعد إزالة طاف، إضافة 1 مل من الايثانول 70٪ لغسل بيليه بواسطة هزاز ببطء وتتحرك بلطف الإيثانول على بيليه عجلت عدة مرات. الإبقاء على الإيثانول في الأنبوب. </li><li style=";text-align:right;direction:rtl"> بعد غسل الأولي، الطرد المركزي العينة لمدة 1 دقيقة في 2،000 x ج و 20 درجة مئوية. ويمكن القيام بهذه الخطوة إما الطرد المركزي في 4 درجات مئوية أو 20 درجة مئوية. وقد تبين أي أثر ملموس للحرارة لهذه الخطوة. </li><li style=";text-align:right;direction:rtl"> بعد غسل الأولي والطرد المركزي من بيليه، صب ببطء غسل الإيثانول من الأنبوب وتجاهل، ثم إجراء غسل الثاني بتكرار الخطوات 5.3 و 5.4. </li><li style=";text-align:right;direction:rtl"> بعد إزالة طاف من غسل الثاني، والسماح بيليه الهواء الجاف لمدة 15 دقيقة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إذا لم تجفف العينة تماما، الهواء الجاف لمدة 15 دقيقة أخرى. </li></ol></li></ol> 6. الإماهة من gDNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مرة واحدة قد جفت عينة، إضافة 300 ميكرولتر من تريس، EDTA لترطيب gDNA بيليه المجففة. </li><li style=";text-align:right;direction:rtl"> دوامة العينة لمدة 5 ثانية على سرعة متوسطة ومكان في65 ° C حمام الماء الساخن لمدة 1 ساعة. </li><li style=";text-align:right;direction:rtl"> إزالة عينات من حمام الماء واحتضان O / N في RT. ملاحظة: جميع المنتجات والكواشف المستخدمة المذكورة في الجدول المواد، وكذلك الجدول 4. </li></ol>

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The authors have nothing to disclose.

الإجراء الحالي هو الأمثل DNA بروتوكول الاستخراج التي قد تحسن بشكل كبير العائد من الحمض النووي الوزن الجزيئي عالية مقارنة بالطرق القياسية، دون المساومة على جودة DNA. كانت الخطوة الحاسمة مع أكبر تأثير على العائد على معظم خطوة 5.2، والذي يتضمن خطوة الطرد المركزي أطول أثناء...

الحصول على الحمض النووي عالية الجودة للدراسات الجينية البشرية أمر أساسي في عملية اكتشاف الجين المرض. الدم، على الرغم تتطلب إجراء الغازية وكونها أكثر تكلفة من جمع اللعاب أيضا، ويحظي لإنشاء خطوط الخلايا خلد كمصدر لانهائي من الحمض النووي، أو iPSCs للدراسات وظيفية، وأحيانا يستخدم الحمض النووي في الدم عند خطوط الخلايا غير متوفرة. ومع ذلك، يتطلب الحصول على الدم فصاد المدربين والدم لديه نصف عمر أقصر من اللعاب 1. DNA من اللعاب هو أقل كلفة وأكثر سهولة الحصول عليها، لأنها يمكن جمعها وإرسالها عبر البريد دون الحاجة إلى فصاد، وبالتالي زيادة برك الموضوع المحتملة إلى ما وراء منطقة مستجمعات المياه من المستشفيات والمختبرات 2. يمكن تحسين التحاق دراسة عندما يكون الخيار من المواضيع إعطاء عينة من اللعاب بدلا من الدم 3، 4. المخاوف بشأن كمية ونوعية الحمض النووي من اللعاب قد تقتصر على نطاق واسع لناه على الرغم من العديد من الدراسات الدراسات الحديثة تظهر مدى ملاءمة اللعاب كله، بمتوسط ​​قدره 4.3 × 10 5 خلية في الملليمتر الواحد، لاختبار الحمض النووي على مدى القديمة مسحات الشدق الأساليب التي لم تحصل على كميات كبيرة من اللعاب 2، 3، 4، 5، 6. في حين أن الأدب متواضع موجود تبين مدى ملاءمة DNA اللعاب كله مستمد للتطبيقات بما في ذلك أساليب التنميط الجيني القائم على ميكروأري 8، 9، 10، وقد درست توجد دراسات الجيل القادم التسلسل (خ ع). كان الهدف لتحسين هذا البروتوكول استخراج الحمض النووي اللعاب كله لتحقيق أقصى قدر من كمية ونوعية لتطبيقات علم الوراثة بطريقة فعالة من حيث التكلفة التي يتم تنفيذها بسهولة في المختبرات المشتركة مع الكواشف والمواد الاستهلاكية. استخراج الحمض النووي من اللعاب يتطلب العديد من الإجراءات: 1) جمع وتخزين، 2) تحلل الخلايا، 3) العلاج ريبونوكلياز، 4) البروتين هطول، 5) هطول الأمطار الإيثانول، 6) الإماهة الحمض النووي. الحل DNA لتحقيق الاستقرار العازلة، وصفت2 سابقا، وظائف كاف دون تغيير. ولم تبذل أية محاولة لتحسين العلاج ريبونوكلياز وDNA الخطوات الإماهة. لكل خطوة المتبقية، وقد تم تحديد العديد من المتغيرات التي يمكن أن تؤثر على الإنتاجية. تم التلاعب بها كل متغير على حدة وتقييم التحسن في المحصول وجودة إحصائيا. للمتغيرات التي عرضت لتحسين العائد و / أو نوعية الحمض النووي، وأدرجت القيم المثلى في البروتوكول النهائي.

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			<td height="21" style="height:21px;">Equipment</td>
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			<td height="43" style="height:43px;width:88px;">Name of Equipment</td>
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			<td height="64" style="height:64px;width:88px;">Analog Vortex Mixer</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">02-215-365</td>
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			<td height="43" style="height:43px;width:88px;">Centrifuge 5810R</td>
			<td style="width:88px;">Eppendorf</td>
			<td style="width:84px;">5811 000.010</td>
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collection and extraction of saliva dna for next generation sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

لتحديد المعايير المثلى لاستخراج الحمض النووي تم تنفيذ سلسلة من عمليات الاستخراج DNA المقترنة. تم تقسيم عينة اللعاب واحدة واختبار كل جزء واحد من اثنين من القيم الممكنة للمتغير معين. وأجريت ثمانية على الأقل مكررات من كل اختبار تقرن (على سبيل المثال، تم aliquoted عينة ال...

Watch this Scientific Journal Video about Collection and Extraction of Saliva DNA for Next Generation Sequencing at JoVE.com

Collection and Extraction of Saliva DNA for Next Generation Sequencing

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

جمع واستخراج الحمض النووي من اللعاب لتسلسل الجيل القادم

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

collection-and-extraction-of-saliva-dna-for-next-generation-sequencing

Research

JoVE Journal

Medicine

38.5K Views.  The Research Institute at Nationwide Children's Hospital. DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Video: Collection and Extraction of Saliva DNA for Next Generation Sequencing

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

جيل من المجهرية الجيني للتطبيقات الطبية الحيوية

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

وα الاختبار: رابيد تعداد CD4 خالية من الخلايا عن طريق اللعاب الجامعة

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

بروتوكول لجمع البنكرياس الإنسان

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into a &#8216;recipient&#8217; block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 &#8216;recipient&#8217; blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

الجيل المقبل من الأنسجة ميكروأري (ngTMA) بروتوكول للدراسات العلامات البيولوجية

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

التكامل بين الرطب والعمليات مقعد الجافة يحسن المستهدفة تسلسل الجيل التالي من ذات جودة منخفضة ومنخفضة كمية التحليلات ورم

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

استخراج الحمض النووي للدياتوم الاقتصادي باستخدام مجموعة معدلة

Extraction of Diatom DNA from Water Samples and Tissues

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

تسليط الضوء على المؤلف: اختبار المشطورة لفحص الغرق الشرعي 

This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

استخراج الحمض النووي للدياتوم من عينات المياه والأنسجة

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...

اكتشاف العلامات الحيوية غير الغازية للبروتينات المسيل للدموع باستخدام LC-MS

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

تسليط الضوء على المؤلف: تطوير تحليل السائل المسيل للدموع باستخدام بروتوكول موحد لأبحاث البروتيوميات 

This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.

تحضير عينة محاصرة بتعليق البروتين للبروتينات المسيل للدموع بواسطة الكروماتوغرافيا السائلة - قياس الطيف الكتلي الترادفي

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...