우리는 기꺼이 그 연구에 사용되는 시약 그들의 관대한 기부에 대한 분자 디바이스에게 분석 기술 / 아크 투 르스을 인정합니다. 우리는 또한 사후 인간 뇌 조직을 제공하는 하버드 조직 리소스 센터 감사 기금 :. RO1MH76060 및 P50MH080272.

<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>

이 비디오 문서의 생산은 생명 기술에 의해 후원되었다.

여기, 우리는 아크 투 르스 XT 시스템과 관련 RNA 추출 키트를 사용하여 이기종 사후 뇌 조직에서 균일한 세포 집단을 추출하는 프로토콜을 사용자 정의하려고 시도합니다. 위에서 설명한대로, 우리는 RNA의 무결성을 극대화하기 위해, 회사가 제공하는 원래의 프로토콜에 몇 가지 수정을 만들었습니다. 단일 세포의 성공적인 촬영은 전적으로 좋은 RNA와 함께 최대의 세포를 달성하기 위해 캡처하기 전에 단계를 최적화에 따라 달라집니다. 이 단계에서는 조직 sectioning 및 얼룩과 관련된 다양한 매개 변수를 포함합니다. 즉, 조직의 준비에 대해서는 이러한은 다음과 같습니다 1), 2 sectioning 때 온도 기울기를 감소) 만 4, 얼음의 모든 탈수 단계를 수행 슬라이드 당 2 섹션 3) 배치) 증가) 얼룩, 5 RNase 억제제를 추가 삼분로 최종 100 % 에탄올의 탈수 기간. 안부가 캡처하기 위해, 우리는 매크로 설정에서 소프트웨어와 CapSure HS 모자,의 조합이 최적의 결과를 보장한다고 생각합니다. 높은 습도는 크게 조직 &quot;엘리베이터&quot;를 감소 등 습도 제어 환경, 캡처 한 세포에 중요합니다. 고온 또한 RNA의 품질에 부정적인 영향을 미칠 수 있습니다. PicoPure 키트와 RNA 격리 프로토콜 동안 두 세척 단계가 있습니다. 그것은 모든 씻어 버퍼가 RNA의 용출에 대한 다른 microcentrifuge 관에 전송하기 전에 제거 확인하는 최종 샘플에있는 버퍼의 존재로 증폭을위한 적은 시작 RNA가 발생합니다 중요합니다. 이것을 보장하기 위해, 우리는 제공된 프로토콜의 제안 2.5 분 대신에 2 분 동안 최종 세척 단계를 원심 분리기. 대부분의 경우, 선형 증폭 프로토콜은 제조 업체의 지시에 따라되었습니다. 이 프로토콜을 사용하면 단 두 개의 중요한 포인트를 고려해야한다 : 1) 튜브 사이의 온도 자전거 타는 사람에 0.5ml 블록과 함께 제공된에만 0.5ml 튜브를 사용하여 샘​​플을 초과하는 전송을 통해 RNA의 손실을 제한하려고 및 2) cDNA의 정화를위한 정화 열 샘플을 전송할 때, 첫 번째 전송 후 샘플 튜브를 스핀 다운해야합니다. 이것은 정화 열의에 추가하고 따라서 cDNA 복구를 향상시킬 수 있습니다 샘플의 추가 1 2ul집니다. 귀하의 제한 아르나 샘플 비오틴 - 라벨을위한 터보 최종 라벨링 키트를 이용하는 경우, 우리는 여분의 몇 μl를 추가하면 표시된 RNA가 열을 추출 수율 증가, 원심 분리 단계 전에 물을있어 좋습니다. 원심 분리의 추가 분 또한 같은 FFPE 조직과 같은 조직을 저하와 작업하는 특히,이 점에서 도움이 될 것입니다. 우리는이 프로토콜은 사용자가 이러한 불일치 사후 뇌 조직 내에 단세포 집단, 작은 균일한 구조를 분리 수 있도록 할 것이라 확신. 이 조사에서 특정 세포 유형으로 대상 결과로 이어지는 두뇌 내의 여러 레이어에 위치 주변의 구조와 다른 세포 타입의 희석 효과를 감소시킵니다. 결과 RNA의 품질이 microarray 연구에 대한 충분합니다, 우리는 질병 상태에 영향을 특정 세포 집단의 특정 분자 서명을 정확하게 시작할 수 있습니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

우리는 레이어 III의 피라미드 뉴런의 유전자 발현 프로필을 심문하여 정신 분열증 환자에서 볼 뛰어난 시간적 이랑에있는 회색 물질 감소를 조사하기 위해 제안. 그것은 잘 대뇌 피질은 다양한 지역, 레이어와 독특한 세포와​​ 분자 작곡 따라서 차동 유전자 발현 프로파일이 특징입니다 각각의 세포 유형을 구성된 매우 이질적인 구조로되어 알려져있다. 조직 이질의 혼란함을 주죠 효과를 회피하기 위해, 우리는 특정 세포 유형 즉 피라미드 뉴런을 분리하기 위해 레이저 캡처 microdissection (LCM)을 사용합니다. Histogene의 얼룩 솔루션 물들일 약 500 피라미드 뉴런은 아크 투 르스 XT LCM 시스템을 사용하여 점령했다. RNA는 다음 캡처 세포로부터 격리 및 아크 투 르스 / 분자 장치 키트를 사용하여 T7 기반 선형 증폭의 두 라운드를 받았습니다했다. Experion LabChip (바이오 래드) 겔과 electropherogram은 microarrays에 필요한 과거 600nt를 확장 성적표 길이와 양질에게 (M) RNA를 지적했다. 2.5 A260/280 비율에 의해 표시로 얻은 mRNA의 양은 허용 의미 샘플 순도와 51μg 평균. 유전자 발현은 Affymetrix의 GeneChip 인간 X3P 프로브 어레이를 사용하여 프로파일되었습니다.

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

우리는 사후에 인간의 두뇌에있는 우수한 시간적 이랑의 레이어 III에서 단일 세포 인구, 피라미드 뉴런에서 RNA를 분리하고 추출 레이저 캡처 microdissection를 사용하여 프로세스를 설명합니다. 우리는 이후 선형 (T7 기준) mRNA를 증폭하고, Affymetrix microarray 인간 X3P에 샘플을 잡종.

인간의 사후 뇌 조직에서 단세포 집단의 고품질 RNA 구하기

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

obtaining-high-quality-rna-from-single-cell-populations-human

Research

JoVE Journal

Biology

30.5K 조회수.  McLean Hospital. 우리는 사후에 인간의 두뇌에있는 우수한 시간적 이랑의 레이어 III에서 단일 세포 인구, 피라미드 뉴런에서 RNA를 분리하고 추출 레이저 캡처 microdissection를 사용하여 프로세스를 설명합니다. 우리는 이후 선형 (T7 기준) mRNA를 증폭하고, Affymetrix microarray 인간 X3P에 샘플을 잡종.

비디오: 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.

Xenopus laevis 여성의 난자를 구하기

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, ...

연구

생물학

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.

인간의 사후 뇌 조직에서 핵의 연결을 격리

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.

마우스 신경 조직에서 단일 셀 Suspensions의 생성

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.

마우스 콘 Photoreceptors에서 단일 셀 석션 레코딩

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.

인간 배아 줄기 세포 인구에서 Hepatocyte 같은 전지의 강력한 생성

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.

높은 처리량 시퀀싱을위한 고품질 염색질 면역 침전의 DNA 템플릿의 생성 (칩 SEQ)

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.

신선 냉동 마우스 뼈에서 고품질 RNA를 생성하는 레이저 캡처 미세 해부 프로토콜

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.

인간 뇌 조직에서 단백질의 다중 반응 모니터링 기반 검출을 이용한 정량적 프로테오믹스 워크플로우

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.

인간 골관절염 무릎 관절에서 조직 수집 및 RNA 추출

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.