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

참고 : 이전 타액 샘플을 제공하기 위해 모든 주제는 전국 어린이 병원에서 사람을 대상으로 치료를위한 가이드 라인을 준수 동의를 통보했다. 1 타액 수집 및 저장 <ol><li> 타액을 수집하기 전에 피사체의 입 피사체가 물로 입을 헹구어 갖는 식사를 피하거나 샘플을 수집하기 전에 30 분 동안 마시는 음식이나 이물질이 없는지 확인합니다. </li><li> 캡 또는 튜브의 내부에 손이 닿지 않도록 확인하는 DNA 안정화 버퍼 (2) 2.5 ㎖로 15 ㎖의 원심 분리기 튜브를 열고 피사체가 완충 용액에 침 2.5 ㎖의 침이있다. 참고 : 타액의 이상 2.5 ml의 수집은 DNA 안정화 버퍼에 샘플의 부족 비율에서 샘플이 저하 될 수 있습니다. 너무 작은 타액을 수집하는 프로토콜에서 예상 수확량을 감소시킬 것이다. 컬렉션 볼륨을 평가하려면, t 측에 번호 그라디언트를 사용그는 튜브. </li><li> 캡을 교체하고, 혼합물을 균질화 될 때까지 반전으로 섞는다. 격렬한 흔들림이 필요하지 않습니다. 단기 기억 또는 장기 저장을위한 4 ° C (&gt; 3 개월)에 대한 RT에서 샘플을 보관합니다. </li></ol> 2 초기 준비 및 세포 용해 <ol><li> 이전 추출을 시작으로, 37 ° C에 수조를 가열하고, 얼음 양동이를 준비합니다. 세 15 ML 원뿔 원심 분리기 튜브는 각 추출 된 샘플에 대해 필요합니다. 세 튜브 세포 단백질 펠렛, 최종 gDNA를 추출하고, 이소프로판올 및 에탄올 상청액을 보유하는 데 사용된다. </li><li> 15 초 후 중간 속도로 소용돌이를 저장에서 샘플 및 반전 샘플을 여러 번 검색합니다. </li><li> 깨끗한 15 ㎖의 원심 분리 관에 시료를 2.5 ㎖에 분배하고, 세포 용해 용액 5 ㎖를 추가합니다. 반전에 의해 시료 50 시간 혼합하고, 30 분 동안 RT에서 배양한다. </li></ol> 3 RNA 제거 <ol><li> 의 RNase 솔의 40 μl를 추가100 ㎎ / ㎖에서의 ution, 그리고 15 분 동안 37 ° C에서 배양한다. </li><li> 3 분 동안 얼음에 37 ° C의 물을 욕조에서 샘플을 제거하고 멋진. <ol><li> 의 RNase 인큐베이션 후, 프로토콜의 DNA의 재수 단계에서 65 ° C로 수조의 온도를 증가시킨다. </li></ol></li></ol> (4) 단백질 및 지질 제거 <ol><li> , ㎖ / 20 밀리그램에 테 K 솔루션의 50 μl를 추가 반전에 의해 여러 번 혼합과 30 분의 최소 RT 부화. 참고 :이 프로토콜에 대한 가능한 일시 정지 포인트입니다. 추출이 완료 될 때까지 K 테 수용액을 첨가 한 후, 샘플을 4 ° C에서 저장 될 수있다. 최대 24 시간 동안 4 ℃에서 보관 추출 수율 또는 DNA의 품질에 상당한 영향을 미치는 것으로 도시되지 않았다. 이 단계에서 장기 저장이 평가되지 않았습니다. </li><li> 10 분 동안 얼음에 20 고속 초, 및 장소를 적극적 소용돌이 단백질 침전 솔루션의 1.7 ML을 추가합니다. </l난&gt; <li> 샘플은 3000 XG에 10 분, 4 ° C에 10 분, 원심 분리기에 얼음에 냉각 한 후에. 침전 된 단백질은 단단한 펠릿이 계속 형성해야합니다. 펠릿을 꽉 없거나 흐린 용액이 여전히 있다면, 샘플은 5 분 더 얼음 위에서 냉각 할 수 있고, 원심 분리를 반복. 샘플은 단단한 펠릿을 보장하기 위해 얼음에 보관해야합니다. </li></ol> (5) 분리 및 gDNA를 정제 <ol><li> 깨끗한 15 ㎖의 원심 분리 관에, 피펫 아이소 프로필 알코올 5 ㎖ 및 20 ㎎ / ㎖에서 순수 글리코겐 솔루션의 8 μL. </li><li> 침전 된 단백질 펠렛을 남겨두고, 아이소 프로필 알코올과 글리코겐 솔루션을 포함하는 튜브에 4.3 단계에서 gDNA를가 포함 된 상층 액을 붓는다. 상층 액을 추가 한 후, 부드럽게 3000 XG 및 4 ℃에서 30 분 동안 샘플을보기 반전 원심 분리에 의해 50 시간 혼합한다. </li><li> 깨끗한 15 ML 튜브에 천천히 뜨는을 붓고. 상청액을 제거한 후, 70 % 에탄올 1 ㎖를 추가 할수회 천천히 부드럽게 흔들 석출 펠렛 위에 에탄올을 이동하여 펠렛을 씻는다. 튜브에서 에탄올을 유지합니다. </li><li> 초기 세척 한 후, 2000 XG에 1 분, 20 ° C에 대한 샘플을 원심 분리기. 이 원심 분리 단계는 4 ° C에서 20 ° C 중 하나에서 수행 할 수 있습니다. 온도의 큰 영향이 단계에서는 도시되지 않았다. </li><li> 펠렛의 초기 세척 및 원심 분리 후, 천천히 튜브에서 에탄올 세척을 부어 버리고, 다음 단계 5.3 및 5.4를 반복하여 두 번째 세척을 수행합니다. </li><li> 두 번째 세척에서 상층 액의 제거 후, ​​15 분간 자연 건조 펠렛을 할 수 있습니다. <ol><li> 시료를 완전히 건조되지 않은 경우, 또 다른 15 분 동안 공기 건조. </li></ol></li></ol> gDNA를의 6 재수 <ol><li> 샘플이 건조되면, 건조 gDNA를 펠렛을 재수하는 트리스 - EDTA (에틸렌 다이아 민 테트라 초산) 300 μl를 추가합니다. </li><li> 중간 속도와 장소에서 5 초 동안 소용돌이 샘플1 시간 동안 65 ° C 온수 목욕입니다. </li><li> 수조에서 샘플을 제거하고 RT에서 O / N을 품어. 참고 : 모든 제품 및 시약 재료 표에 나와있는 사용뿐만 아니라, 표 4. </li></ol>

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The authors have nothing to disclose.

본 절차는 상당히 DNA의 품질을 손상시키지 않고, 표준 방법에 비해 고 분자량 DNA의 수율이 향상되었다 최적화 DNA 추출 프로토콜이다. 수율을 최대한에 가장 큰 효과 중요한 단계 널리 11을 배포하지 않은 일을 제외하고, 여기에서 검토 게시 된 프로토콜에 비해 에탄올 침전 동안 이상 원심 분리 단계를 포함하는 단계 5.2였다. 이 이상 원심 분리와 관련된 DNA 품질의 변경은 전체 타액 컬렉션...

인간 유전 연구 고품질 DNA를 얻는 것은 질병 유전자 발견 프로세스에 필수적이다. 혈액, 타액 수집보다 더 비싼 또한 침략적인 절차를 요구하고 있지만, 기능 연구에 대한 무한 DNA의 소스 또는 iPSCs로 불후의 세포 라인을 만드는 선호하고, 세포주를 사용할 수없는 경우 때때로 혈액 DNA가 사용됩니다. 그러나, 혈액을 획득하는 훈련 된 phlebotomist을 필요로 피가 침 하나보다 짧은 반감기를 가지고있다. 그것을 수집함으로써 잘 병원 실험실 (2)의 집수 영역을 넘어 전위 피사체 풀을 증가 phlebotomist 필요없이 이메일을 통해 전송 될 수 있기 때문에 타액으로부터 DNA는 얻을 저렴하고 용이하다. 과목 대신 혈액 3, 4의 타액 샘플을 채취 할 수있는 옵션이있을 때 연구 등록이 개선 될 수있다. 수량과 타액에서 DNA의 질에 대한 우려는 우리가 널리 제한되어 있습니다타액 2, 3, 4, 5의 상당한 양을 구하는 않았다 이전 협측 면봉 방법을 통해 DNA 테스트에 대한 밀리리터 당 4.3 × 105 세포의 평균으로, 전체 타액의 적합성을 나타내는 수많은 연구 최근의 연구에도 불구하고, 즉, 6. 겸손한 문학 마이크로 어레이 기반 방식 팔을 포함하여 유전자형 응용 프로그램에 대한 전체 타액 유래 DNA의 적합성을 보여주는 존재하지만 9, 10, 더 연구가 시도되지 않은 차세대 시퀀싱 (NGS). 이 모든 타액 DNA 추출 프로토콜을 최적화하는 목적은 쉽게 일반적인 시약 및 소모품으로 실험실에서 구현 비용 효과적인 방식으로 응용 프로그램에 대한 유전학 수량 및 품질을 최대화하는 것이다. 타액에서 DNA 추출은 여러 절차가 필요합니다 : 1) 수집 및 저장, 2) 세포 용해, 3)의 RNase 처리, 4) 단백질 침전, 5) 에탄올 침전, 6) DNA의 수분 보충을. 기재된 DNA 안정화 버퍼 용액이전에이 적절하게 변경없이 작동합니다. 의 RNase 처리 및 DNA의 수분 보충 단계를 최적화하기위한 어떠한 시도도하지 않았다. 나머지 각 단계의 수율에 영향을 미칠 수있는 여러 변수가 확인되었다. 각 변수를 개별적으로 조작하고, 수율 및 품질의 향상이 통계적으로 평가 하였다. 수율 및 / 또는 DNA의 품질을 향상시키기 위해 도시 된 변수의 최적 값은 최종 프로토콜에 포함되었다.

<table border="0" cellpadding="0" cellspacing="0" width="260">
	<tbody>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">15ml Centrifuge Tubes</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">12-565-268</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Cell Lysis Solution</td>
			<td style="width:88px;">Qiagen</td>
			<td style="width:84px;">158908</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Proteinase K</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:84px;">P6556</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">Protein Precipitation Solution</td>
			<td style="width:88px;">Qiagen</td>
			<td style="width:84px;">158912</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Isopropanol</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">A416-4</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Glycogen</td>
			<td style="width:88px;">EZ-BioResearch</td>
			<td style="width:84px;">S1003</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">70% Ethanol</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">04-355-305</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Tris-EDTA (TE)</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP2473-1</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">NaCl</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">AC194090010</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Tris HCl</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP1757-100</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">EDTA(0.5M) Solution</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">03-500-506</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">Sodium Dodecyl Sulfate</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP166-100&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:88px;">Name of Equipment</td>
			<td style="width:88px;">Distributor</td>
			<td style="width:84px;">Catalog#</td>
		</tr>
		<tr height="64">
			<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>
		</tr>
		<tr height="43">
			<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>
		</tr>
	</tbody>
</table>

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.

DNA는 한 쌍의 DNA를 추출하는 일련의이 수행 된 추출을위한 최적의 매개 변수를 결정합니다. 하나의 타액 샘플을 분할하고 각 부분은 주어진 변수에 대한 두 가지 값 중 하나를 사용하여 테스트되었습니다. 각 쌍 테스트 적어도 8 회 반복 (예를 들어, 하나의 타액 샘플과 초기 50 ° C에서 배양없이 모두 추출을 테스트하기 위해 분주했다)을 시행 하였다. 총 DNA 수율, 280 분의 260 값 230분의 260 값...

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.

차세대 시퀀싱에 대한 수집 및 타액 DNA의 추출

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

38.8K 조회수.  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.

비디오: 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.

바이오 메디컬 응용 프로그램을위한 알긴산 Microspheres의 생성

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

연구

의학

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.

α-테스트 : 전체 타액을 사용하여 빠른 셀 프리 CD4의 열거

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.

인간의 췌장을위한 컬렉션 프로토콜

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.

바이오 마커 연구 차세대 조직 마이크로 어레이 (ngTMA) 프로토콜

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.

낮은 품질과 낮은 수량 종양 생검의 습식 및 건식 벤치 프로세스의 통합을 대상으로 최적화하여 차세대 시퀀싱

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

Modified Kit를 사용한 경제적인 규조류 DNA 추출

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.

저자 스포트라이트: 법의학 익사 검사를 위한 규조류 검사 

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

물 샘플 및 조직에서 규조류 DNA 추출

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

LC-MS를 사용한 눈물 단백질체학을 위한 비침습적 바이오마커 발견

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.

저자 스포트라이트: 단백질체학 연구를 위한 표준화된 프로토콜을 사용한 눈물액 분석 발전 

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.

액체 크로마토그래피-탠덤 질량분석법에 의한 눈물 단백질체학을 위한 단백질 현탁액 포획 시료 전처리

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