이 작품은 당뇨병 캐나다에 의해 지원 되었다.

<ol>
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T., Eskenazi, B., Bastaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+sample+collection+and+processing+for+molecular+epidemiological+studies.">Biological sample collection and processing for molecular epidemiological studies.</a> Mutat Res. 543 (3), 217-234 (2003).</li><li>Ibrahim, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+and+visceral+adipose+tissue:+structural+and+functional+differences.">Subcutaneous and visceral adipose tissue: structural and functional differences.</a> Obes Rev. 11 (1), 11-18 (2010).</li><li>Mann, A., Thompson, A., Robbins, N., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization,+identification,+and+excision+of+murine+adipose+depots.">Localization, identification, and excision of murine adipose depots.</a> J Vis Exp. (94), (2014).</li><li>Hemmrich, K., Denecke, B., Paul, N. E., Hoffmeister, D., Pallua, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Isolation+from+Adipose+Tissue:+An+Optimized+Procedure+for+High+RNA+Yield+and+Integrity.">RNA Isolation from Adipose Tissue: An Optimized Procedure for High RNA Yield and Integrity.</a> Laboratory Medicine. 41 (2), 104-106 (2010).</li><li>Cirera, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+method+for+isolation+of+total+RNA+from+adipose+tissue.">Highly efficient method for isolation of total RNA from adipose tissue.</a> BMC Res Notes. 6, 472 (2013).</li><li>Tan, S. C., Yiap, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA,+RNA,+and+protein+extraction:+the+past+and+the+present.">DNA, RNA, and protein extraction: the past and the present.</a> J Biomed Biotechnol. 2009, 574398 (2009).</li><li>Sellin Jeffries, M. K., Kiss, A. J., Smith, A. W., Oris, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+commercially-available+automated+and+manual+extraction+kits+for+the+isolation+of+total+RNA+from+small+tissue+samples.">A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples.</a> BMC Biotechnol. 14, 94 (2014).</li><li>Tan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+prorenin/renin+receptor+on+the+development+of+obesity+and+associated+cardiometabolic+risk+factors.">Impact of the prorenin/renin receptor on the development of obesity and associated cardiometabolic risk factors.</a> Obesity (Silver. Spring). 22 (10), 2201-2209 (2014).</li><li>Taylor, S., Wakem, M., Dijkman, G., Alsarraj, M., Nguyen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+RT-qPCR-Publishing+data+that+conform+to+the+MIQE+guidelines.">A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines.</a> Methods. 50 (4), 1-5 (2010).</li><li>Aranda, P. S., LaJoie, D. M., Jorcyk, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleach+gel:+a+simple+agarose+gel+for+analyzing+RNA+quality.">Bleach gel: a simple agarose gel for analyzing RNA quality.</a> Electrophoresis. 33 (2), 366-369 (2012).</li><li>Mercure, C., Prescott, G., Lacombe, M. J., Silversides, D. W., Reudelhuber, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+increases+in+circulating+prorenin+are+not+associated+with+renal+or+cardiac+pathologies.">Chronic increases in circulating prorenin are not associated with renal or cardiac pathologies.</a> Hypertension. 53 (6), 1062-1069 (2009).</li><li>Dusaulcy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-specific+disruption+of+autotaxin+enhances+nutritional+fattening+and+reduces+plasma+lysophosphatidic+acid.">Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.</a> J. Lipid Res. 52 (6), 1247-1255 (2011).</li><li>Nakamura, K., Fuster, J. J., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipokines:+a+link+between+obesity+and+cardiovascular+disease.">Adipokines: a link between obesity and cardiovascular disease.</a> J Cardiol. 63 (4), 250-259 (2014).</li><li>Taylor, S. C., Mrkusich, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+RT-quantitative+PCR:+firsthand+observations+of+implementation+of+minimum+information+for+the+publication+of+quantitative+real-time+PCR+experiments+(MIQE).">The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE).</a> J Mol Microbiol Biotechnol. 24 (1), 46-52 (2014).</li><li>Hummon, A. B., Lim, S. R., Difilippantonio, M. J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+solubilization+of+proteins+after+TRIzol+extraction+of+RNA+and+DNA+from+patient+material+following+prolonged+storage.">Isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from patient material following prolonged storage.</a> Biotechniques. 42 (4), 467-470 (2007).</li></ol>

저자는 공개 없다.

다음 먹이 HF-다이어트, 비만 쥐 몸 및 백색 지방 조직 가중치 N 규정식 먹인 쥐에 비해 증가을 발견 했다. 페 놀 솔루션을 사용 하 여 추출 하는 RNA 좋은 순도와 샘플을 굴복. Leptin은 주로 지방 조직에 의해 생산 하는 adipokine 및 지방 대량16와 긍정적으로 상관 관계 알려져 있다. 예상 했던 대로, leptin mRNA 식 그들의 뚱뚱한 질량을 가진 concomitance에서 비만 생쥐에서 증가 했다.
<p cl...

비만 타입-2 당뇨병1등 합병증으로 이어질 수 있는 전 세계적으로 유행 하는. 다이어트 유발 된 비만 유전자 변형 동물 모델에 비만 관련 된 합병증의 연구 자주 사용 됩니다. 전통적으로, 백색 지방 조직 초과 에너지를 위한 저장 격실으로 알려져 있다 이며 주로 지질 구성 된 갈색 지방 조직 열2,3로 에너지를 변환 하는 동안. 지방 조직 동적 확장 하 고 음식 섭취 량과 신체 활동 등 많은 요인에 따라 축소 됩니다. 따라서, 이러한 변화에 기여 요인 결정, 적절 한 지방 조직 수집 및 처리는 필요4.
가운데 흰색 adipose 조직, 그것 일반적으로 피하와 내장 지방 저장소 해부학 지역화 등 다른 속성 있고,2,5기능 허용 됩니다. 따라서, 충돌 하는 결과 또는 큰 변화를 방지 하려면 주의 지방 패드를 수집 하는 때이 다른 지방 저장소 간의 교차 오염을 방지로 이동 해야 합니다.
또한, RNA 또는 단백질 흰 쥐의 지방 조직에서 분리 하는 경우 세 가지 주요 과제 있다. 첫째, 비만 쥐에서 지방 패드를 수집 하지 않습니다 쉬운 일 다른 백색 지방 창 고를 분리 하는 테두리는 항상 명확 하 고, 신장과 심장6등 다른 장기와 달리. 둘째, 지방 조직, RNA 또는 단백질 격리 중의 높은 지질 내용 때문에 지질 층 위에 수레 하 고 샘플에 대 한 직접 액세스를 방지. 셋째, 갈색 지방이 많은 직물, 또는 다른 조직, 반대로 백색 지방 조직 내용이 상당히 낮은 RNA와 단백질 및이 젊은 마우스를 사용할 때 주요 관심사의, 쥐 먹이 정상 (N) 다이어트 이며 쥐 것으로 예상 된다 낮은 뚱뚱한 질량 (즉 코 모델, 치료 약물, 운동 훈련, 등.) 7 , 8.
따라서, 지방 조직에서 RNA를 분리 하는 적절 한 방법을 선택이 중요 합니다. 페 놀/클로 프롬 추출 대체 방법을 상용 키트 있습니다. 그들은 일반적으로 RNA 정화 열9에 따라 초기 페 놀 추출 단계를 기반으로 합니다. 그러나, 그 키트는 일반적으로 더 비싼 및 샘플 낮은 수율의 RNA 품질 변수 수도 주지만 적은 시간이 소요 됩니다. 그러나, 여기에 설명 된 페 놀 솔루션/클로 프롬 추출에 가장 큰 장점 중 하나는 RNA, DNA, 그리고 단일 샘플10에서 단백질 분리의 가능성입니다. 쥐 지방 패드는 일반적으로 작은 있기 때문에 (특히 상체 마우스 모델)에서 RNA와 단백질의 소량을 주고, 이러한 프로토콜 작은 샘플에서 얻을 수 있는 데이터를 극대화 합니다.
이 문서의 목적은 3 쥐 백색 지방 조직 창 고 수량 및 RNA 격리의 품질에서 적절 한 샘플 컬렉션을 확인 하는 방법을 자세히 설명 하기 위해. 이 프로토콜에 따라 얻은 RNA 실시간 PCR 분석 실험을 수행 하기 위해 사용할 수 있습니다. 이 프로토콜 교양된 기본 adipocytes에서 RNA 격리에 대 한 것이 아닙니다.

<table><tbody><tr><td>1 mL seringes</td><td></td><td></td><td></td></tr><tr><td>1X TE solution (10 mM Tris-HCl and 1 mM EDTA&amp;bull;Na&lt;sub&gt;2&lt;/sub&gt;. pH 8.0)</td><td></td><td></td><td></td></tr><tr><td>22 G needles</td><td></td><td></td><td></td></tr><tr><td>26 G needles</td><td></td><td></td><td></td></tr><tr><td>75% Ethanol</td><td></td><td></td><td></td></tr><tr><td>Block heater (dry bath)</td><td></td><td></td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C2432-500mL</td><td></td></tr><tr><td>dATP&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Thermo scientific</td><td>R0141</td><td></td></tr><tr><td>dCTP&amp;nbsp;&amp;nbsp;</td><td>Thermo scientific</td><td>R0151</td><td></td></tr><tr><td>dGTP&amp;nbsp;&amp;nbsp;</td><td>Thermo scientific</td><td>R0161</td><td></td></tr><tr><td>DNase I (1 U/&amp;micro;l)&amp;nbsp;</td><td>Thermo scientific</td><td>EN0521</td><td></td></tr><tr><td>dTTP&amp;nbsp;</td><td>Thermo scientific</td><td>R0171</td><td></td></tr><tr><td>Faststart Universal SYBR green Master (Rox)</td><td>Roche</td><td>4913922001</td><td></td></tr><tr><td>Faststart universal SYBR green master (Rox) fluorescent dye&amp;nbsp;</td><td>Roche</td><td>4913914001</td><td></td></tr><tr><td>Filtered tips</td><td></td><td></td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Instrumentarium</td><td>HB275</td><td></td></tr><tr><td>Gauze</td><td></td><td></td><td></td></tr><tr><td>Hammer</td><td></td><td></td><td></td></tr><tr><td>High fat rodent diet</td><td>Bio-Serv, Frenchtown, NJ</td><td>F3282</td><td></td></tr><tr><td>Isopropanol&amp;nbsp;</td><td>Laboratoire MAT</td><td>IH-0101</td><td></td></tr><tr><td>Leptin forward PCR primer (5&amp;rsquo;-GGGCTTCACCCCATTCTGA-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>Leptin reverse PCR primer (5&amp;rsquo;-GGCTATCTGCAGCACATTTTG-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Maxima Reverse Transcriptase (enzyme and 5x buffer)</td><td>Thermo scientific</td><td>EP0742</td><td></td></tr><tr><td>Nanopure water (referred as ultrapure water)</td><td></td><td></td><td></td></tr><tr><td>Nitrile examination gloves</td><td></td><td></td><td></td></tr><tr><td>Nitrile gloves</td><td></td><td></td><td></td></tr><tr><td>Normal rodent diet</td><td>Harlan Laboratories, Madison, WI</td><td>Harlan 2018</td><td></td></tr><tr><td>P1000 pipetman</td><td></td><td></td><td></td></tr><tr><td>P2 pipetman</td><td></td><td></td><td></td></tr><tr><td>P20 pipetman</td><td></td><td></td><td></td></tr><tr><td>P200 pipetman</td><td></td><td></td><td></td></tr><tr><td>Phenol solution (TRIzol)&amp;nbsp;</td><td>Ambion Life Technologies</td><td>15598018</td><td></td></tr><tr><td>Pre-identified aluminium foil</td><td></td><td></td><td></td></tr><tr><td>Quartz spectrophotometer cuvette</td><td></td><td></td><td></td></tr><tr><td>Rack for PCR tube strips</td><td></td><td></td><td></td></tr><tr><td>Racks for RT-PCR tube strips</td><td></td><td></td><td></td></tr><tr><td>Random hexamers&amp;nbsp;</td><td>Invitrogen</td><td>58875</td><td></td></tr><tr><td>Real-time PCR Rotor Gene system&amp;nbsp;</td><td>Corbett research</td><td>RG-3000 Rotor-Gene thermal cycler</td><td></td></tr><tr><td>Refrigerated bench-top centrifuge</td><td></td><td></td><td></td></tr><tr><td>RiboLock RNase Inhibitor&amp;nbsp;</td><td>Thermo scientific</td><td>EO0381</td><td></td></tr><tr><td>RNase-free 1.5 mL eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>RNase-free 1.5 mL screw cap tubes</td><td></td><td></td><td></td></tr><tr><td>RNase-free PCR tube strips (0.2 mL) and caps</td><td></td><td></td><td></td></tr><tr><td>RNase-free water&amp;nbsp;</td><td>Hyclone</td><td>SH30538.02</td><td></td></tr><tr><td>RT-PCR machine</td><td>Qiagen</td><td>Rotor-Gene Corbett 3000</td><td></td></tr><tr><td>RT-PCR tube strips (0.1 mL) and caps</td><td></td><td></td><td></td></tr><tr><td>S16 forward PCR primer (5&amp;rsquo;-ATCTCAAAGGCCCTGGTAGC-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>S16 reverse PCR primer (5&amp;rsquo; ACAAAGGTAAACCCCGATCG-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td>Biochrom</td><td>Ultrospec 3100 pro</td><td></td></tr><tr><td>Stainless steel mortar and pestle</td><td></td><td></td><td></td></tr><tr><td>Surgical pads</td><td>Home made a foam board wrapped in a disposable absorbent underpad</td><td></td><td></td></tr><tr><td>Surgical scissors&amp;nbsp;</td><td>Intrumentarium</td><td>130.450.11</td><td></td></tr><tr><td>Thermal cycler</td><td></td><td></td><td></td></tr><tr><td>Thermal cycler&amp;nbsp;</td><td>Biometra</td><td>Thermocycler</td><td></td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr><tr><td>Weighing spatula</td><td></td><td></td><td></td></tr></tbody></table>

mouse adipose tissue collection and processing for rna analysis

다른 조직에 비해, 백색 지방 조직 있으므로 주로 지질 상당히 적은 RNA와 단백질 콘텐츠를 실시간 PCR 및 서쪽 오 점, 같은 다운스트림 응용 프로그램에 대 한 있다. 추가 단계는이 지질을 방지 하는 데 필요한 지방 조직 샘플에서 RNA 절연은 또한 도전 이다. 여기, 우리 3 해부학 다른 흰색 adipose 조직 생쥐, 이러한 샘플을 처리 하 고 RNA 격리 수행에서 수집 하는 절차를 제시. 우리는 더 실시간 PCR를 사용 하 여 cDNA 그리고 유전자 식 실험의 합성을 설명 합니다. 이로써 설명된 프로토콜에서 동물의 머리와 지방 패드 교차 오염을 조직 수집 하는 동안 다른 지방 패드 사이 혈액 오염의 감소 수 있습니다. 그것은 또한 적절 한 수량 및 RNA 추출의 품질을 보장 하기 위해 최적화 되었습니다. 이 프로토콜 지방 조직 샘플은 실시간 PCR 등 일상적인 실험에 필요한 모든 마우스 모델에 널리 적용 될 수 있지만 기본 adipocytes 세포 배양에서 격리에 적합 하지 않습니다.

검 시 절차에 따라 3 개의 흰색 adipose 조직 수집 되었고 쥐 (N와 HF 다이어트 먹이 쥐)의 두 그룹에서가 중. 예상 했던 대로, 쥐 HF 다이어트 최종 몸 무게와 체중 증가 littermates N 다이어트 (표 1)에 비해 증가 했다. 이러한 관측 함께 했다 이상 2-fold 증가 비만 생쥐에서 PGF, PRF, SCF의 무게에 그에 비해 N 다이어트에.
격리 ...

Watch this Scientific Journal Video about Mouse Adipose Tissue Collection and Processing for RNA Analysis at JoVE.com

Mouse Adipose Tissue Collection and Processing for RNA Analysis

마우스 지방 조직 수집 및 RNA 분석에 대 한 처리

이 문서의 목적은 마우스에서 다른 흰색 adipose 조직 수집, 지방 샘플 처리 RNA를 추출 하는 단계별 절차를 제시 하는 것입니다.

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

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JoVE Journal

Biochemistry

48.2K 조회수.  Université de Montréal. 이 문서의 목적은 마우스에서 다른 흰색 adipose 조직 수집, 지방 샘플 처리 RNA를 추출 하는 단계별 절차를 제시 하는 것입니다.

비디오: Mouse Adipose Tissue Collection and Processing for RNA Analysis

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

총 RNA의 분리 및 다운 스트림 차세대 시퀀싱 및 유전자 발현 프로 파일링에 대한 개별 마우스 뇌 영역의 Microdissection 아닌 레이저 캡처 현미경 접근법

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

연구

신경과학

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

단세포 게놈 응용을 위한 지방 조직 핵의 격리

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

유전학

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

레이저 포획 미세 절에 의해 마우스 척수 모터 신경 세포 체에서 전사 분석을위한 RNA의 생산

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of arthritis research, many studies are focused on inflamed joints that are composed of a complex mixture of bone, cartilage, muscle, stromal cells and immune cells. Here, we established a reliable and robust mechanical method to disrupt inflamed mouse paws into homogeneous pulverized samples in a cryogenically controlled environment. Protein and RNA lysates were processed to enable proteomic and transcriptional endpoints and molecular characterization of relevant disease pathways in local tissue.

A cryogenic pulverization method to process murine paws using a liquid nitrogen freezer mill was developed to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses.

콜라겐 유도 관절염 모델에서 조직 염증의 분자 경로를 평가하기 위한 마우스 발 처리를 위한 저온 분쇄 프로토콜

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of ...

면역학 및 감염병학

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

다운스트림 분석을 위한 냉동 설치류 뇌 영역 모음

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

마우스 췌장에서 RNA 격리 : 리보 뉴 클레아 풍부한 조직

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

생물학

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

RNA 분석 및 대식세포 표현형에 적합한 인간 내장 지방 조직에서 생존 가능한 지방 세포 및 기질 혈관 분획 분리

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

의학

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

유전자 발현 및 조직 학적 분석을위한 단일 마우스 골격근의 연속 지역의 냉동 절편

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

레이저 캡처 서 유전자 표정 분석에 대 한 마우스 눈에서 높은 순수 배수 채널의

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...