Dr. Terri Schochet bu belgede yer alan resimler için hücre kültürlerinin sağlamak için, hücre kültürleri korumak için kaplama ve Kevin Miyashiro için teşekkür ederiz. Buna ek olarak, Kevin Miyashiro, Dr. Peter Buckley ve Tiina Pertiz Arna prosedürü giriş için teşekkür ederiz. Bu çalışma için fon Ulusal Yaşlanma Enstitüsü, Ulusal Ruh Sağlığı ve Pennsylvania Topluluğu İnsan Kaynakları Gerçek Bulucu fonları Enstitüsü oldu.

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E., Andrews, C., Miyashiro, K., McIntosh, T., Becker, K., Barrett, T., Hinkle, D., Dent, G., Marciano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+expression+analysis+of+tissue+sections+and+single+cells.">mRNA expression analysis of tissue sections and single cells.</a> The Journal of Neuroscience. 21, 8310-8314 (2001).</li><li>Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc. Natl. Acad. Sci. USA. 87, 1663-1667 (1990).</li><li>Miyashiro, K., Dichter, M., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+nature+and+differential+distribution+of+mRNAs+in+hippocampal+neuritis:+Implications+for+neuronal+functioning.">On the nature and differential distribution of mRNAs in hippocampal neuritis: Implications for neuronal functioning.</a> Proc. Natl. Acad. Sci. USA. 91, 10800-10804 (1994).</li><li>Sul, J., Wo, C. K., Zeng, F., Jochems, J., Lee, M. T., Kim, T. K., Peritz, T., Buckley, P., Cappelleri, D. J., Maronski, M., Kim, M., Kumar, V., Meaney, D., Kim, J., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+transfer+produces+a+predictable+cellular+phenotype.">Transcriptional transfer produces a predictable cellular phenotype.</a> Proc. Natl. Acad. Sci. USA. 106, 7624-7629 (2009).</li><li>Sow, F. B., Gallup, J. M., Sacco, R. E., Ackerman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+revisited+as+a+tool+for+transcriptomic+analysis:+Application+of+an+excel-based+qPCR+preparation+software+(PREXCEL-Q).">Laser capture microdissection revisited as a tool for transcriptomic analysis: Application of an excel-based qPCR preparation software (PREXCEL-Q).</a> Int. J. Biomed. Sci. 5, (2010).</li><li>Vandewoestyne, M., Deforce, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+forensic+research:+a+review.">Laser capture microdissection in forensic research: a review.</a> Int J Legal Med. 124, (2010).</li><li>Eberwine, J., Bartfai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptomics+of+hypothalamic+warm+sensitive+neurons+that+control+core+body+temperature+and+fever+response+Signaling+asymmetry+and+an+extension+of+chemical+neuroanatomy.">Single cell transcriptomics of hypothalamic warm sensitive neurons that control core body temperature and fever response Signaling asymmetry and an extension of chemical neuroanatomy.</a> Pharmacol. Ther. , (2010).</li></ol>

Dr Eberwine Arna patent Dr Eberwine bir pay sahibi olduğu LBS teknolojileri lisanslı bir mucit.

Notlar ve Sorun Giderme <ul><li> Öncesi ve sonrası hedef hücreyi izole olduğunu gösteren görüntüler ve kalan penumbra bakir kaldı almak isteyebilirsiniz. </li><li> Hasat gibi çok fazla çözüm pipet giriyor, 3 Luer-Lock parçaları yolu ve pozitif basınçlı tüp tutmak için şırınga pistonu kullanabilirsiniz. İstenilen ses işleme türüne göre değişir ama çoğu uygulama için en fazla 5 ul iyi olmalıdır. </li></ul> Moleküler biyoloji teknikleri hakkında genel ipuçları <ul><li> Vortex tüm hisse senedi tüpleri ve bir tepki eklemeden önce onları aşağı döndürün. ASLA vorteks enzimler. </li><li> Reaksiyonları, tampon uygun konsantrasyonda olmasını sağlamak için enzim eklemeden önce iyice karıştırın. </li><li> Mümkünse stratacooler kullanarak -20 ° C&#39;de enzim stokları tutun. Aksi halde, kullanımdan önce doğru kaldırmak buz üzerinde tutmak ve -20 ° C&#39;ye kadar hemen dönmek </li><li> Her zaman pipetleme hataları azaltmak için birden fazla reaksiyon hazırlarken ana karışımları olun. Örneklerin sayısına göre gerekli hacmi hesaplayın ve% 10-20 ekleyin. </li><li> Mağaza RNA -80 ° C geciktirir bozulması. RNA asidik koşullarda oluşur RNA geciktirir apurination tampon depolanan en istikrarlı. </li></ul> Arna Usul Genel Anahat Şekil 4A Arna prosedürün ilk turda göstermektedir. T7-oligo (dT) astar poli-T kısmı, ilk iplikli tepki olarak, Polya kuyrukları bağlanarak mRNA türleri (uzun beyaz dikdörtgen) seçer. Ayrıca bazı microRNA polyadenylated ve bu yordamı Çekilecek. Ancak, daha da önemlisi, hücre içinde en bol RNA, ribozomal RNA&#39;lar, olmaz. Bu oligo mRNA bir şablon olarak kullanarak tamamlayıcı iplikçik cDNA (uzun gri dikdörtgen) sentezlemek için Reverse Transcriptase için bir astar olarak davranır. T7, T7-oligo (dT) astar bölümü başlangıç ​​mRNA dizisi antisens çerçeve T7 RNA polimeraz organizatörü içermektedir. Bu, daha sonra in vitro transkripsiyon tepki olarak kullanılır. Daha sonra, önceki adımda oluşturulan mRNA / DNA hibrid mRNA gecikmeli iplikçik DNA sentezi oluşturulan Okazaki parçaları benzer RNA &quot;primerler&quot; (küçük beyaz dikdörtgenler) oluşturmak Rnase H tarafından kısmen hidrolize olur. DNA Polimeraz I, RNA parçaları, bir şablon olarak mRNA tamamlayıcı DNA kullanılarak asal DNA sentezi için kullanır. &#39;3&#39; sonraki RNA parçası, 5 ulaştığında nükleaz faaliyet ribonucleotides kaldırır ve deoxyribonucleotides ile değiştirir. DNA ligaz önde gelen iplikçik yedek tam olmayan herhangi bir ipliklerini Arter eklenir. T4 DNA Polimeraz RNA parçaları, DNA Polimeraz I oluşturarak ilk primerler olarak nerede görev alanları doldurmak için eklenen bir künt uçlu, çift tıklatın IVT reaksiyon gerçekleştirmeden önce arıtılarak cDNA mahsur. IVT tepki olarak, T7 RNA polimeraz anlamda iplikli bir şablon olarak kullanarak çift cDNA telli ve sentezler antisens RNA molekülleri (uzun siyah dikdörtgenler) dahil T7 organizatörü bağlanır. Bu binlerce antisens RNA molekülleri her çift zincirli cDNA molekülü (Şekil 4A) üretildiği amplifikasyon adım olarak hizmet vermektedir. Şekil 4B tasvir gibi ikinci turda, başlangıç ​​RNA bu yana ilk turda biraz daha farklı bir ters transkripsiyon reaksiyonu ile başlar antisens (düz siyah dikdörtgen) ve T7-oligo tarafından hedef alındı ​​polyadenylated kuyruk yoksun (dT) ilk turda astar. Bu nedenle, bu reaksiyon rastgele primerler (küçük gri dikdörtgenler) ve daha sonra denatüre RNA ile astarlanmalıdır. Ikinci iplikçik reaksiyon sonra önceki ters transkripsiyon tepki olarak oluşturulan anlamda RNA&#39;nın 3 &#39;ucuna poli-dizisi bağlanır T7-oligo (dT) astar ile astarlanmalıdır. Başka bir IVT reaksiyon ilk turda olduğu gibi aynı şekilde yapılır. Bu ikinci turda genellikle, tek bir hücre üç tur amplifikasyon elde etmek için en az bir kez tekrarlanır. Uygulamalar Biz bu yazıda olduğu teknikleri, uygulamalar çok sayıda tercüme edilebilir. Tek hücre izolasyonu protokol akut dilimler halinde kullanılmak üzere modifiye edilebilir. 14 teknik olarak daha zor olsa da bu alternatif hazırlanması, aynı ilkeler geçerlidir. Ayrıca, pipet boyutu biraz ayarlanır ise hücrelerin fizyolojisi kayıtları hasat fizyolojik çıkışları arkasında moleküler mekanizmaları iyi kontrollü bir soruşturma için izin vermeden önce yapılmış olabilir. Başka bir hafif bir değişiklik hücre soma süreçleri izole etmektir. Bu uygulama için 15, bir pipet ile hücre gövdeleri toplamak ve daha sonra yeni bir pipet yardımıyla geri dönün ve 100-300 tespit dendrit toplamakes veya toplama tüpünün başına aksonlar 16 Bir kez hücreler, aynı hücre popülasyonlarının içinde bile farklı ve karşılaştırmalar mRNA bollukları ve kompozisyonlar arasında yapılabilir, hasat edilmiştir. Üçüncü turda Arna içine biotinlenmiş-UTP birleştirilerek bu göreceli mRNA bollukları belirlemek için mikroarray analizi sağlar. Orijinal mRNA nüfusun kompozisyonu da yeni nesil sıralama kullanarak Arna işlemden sonra tespit edilebilir. Güçlendirilmiş Arna da, bir hücre tipi mRNA&#39;ların tam bir set, ikinci hücre tipi fenotipi eski haline geçiş ikna etmek için farklı bir hücre tipi transfekte olduğu hücre fenotipi dönüşüm çalışmaları onaylamak için kullanılabilir bir prosedür laboratuvarı tarafından geliştirilen ve TIPeR olarak bilinen 17 Bu çalışmalar, hastalık durumları ve hücre fenotipleri ve bu tür çalışmaların eğitim için yararlıdır laboratuvarda halen devam etmektedir . RT-PCR veya qPCR hücre spesifik genlerin ifadesi onaylamak için güçlendirilmiş malzeme üzerine yapılabilir. Ayrıca, transfeksiyon veya transdüksiyon verimliliği tek hücre düzeyinde değerlendirmeler yapılabilir. Avantajları ve Sınırlamalar Soyut olarak belirtildiği gibi, analiz için tek hücre izolasyonu, heterojen hücre popülasyonlarının analizi ile görülen ortalama etkileri ortadan kaldırır. Bu ortalama etkileri tek bir hücre içinde bol transkript temsil eden ve ortalamayla çok düşük bolluğu transkript tespiti ve önlenmesi ile mRNA bollukları yanlış. Flow sitometri hücreleri tek tek sıralamak için kullanılan, ancak bu yöntem hücre spesifik belirteçler ve pahalı ekipman bilgisi gerektirir. 18 Lazer yakalama mikrodiseksiyon UV veya IR lazer yakalama mikrodiseksiyon sistemleri ile ya da hatta tek bir hücre ve hücre içi çekim için izin ancak ilgi hücreleri gerektirir çok ince kesitler yüzeyinde bulunan 19 flow sitometri benzer, lazer yakalama mikrodiseksiyon da pahalı ekipman gerektirir. Yukarıda açıklanan elektrot tabanlı toplama tekniğinin en önemli avantajlarından biri, değerli elektrofizyolojik veriler aynı hücre üzerinde yapılacak işlevsel ve transcriptome analiz için izin, hasattan önce ilgi hücre elde edilebilir 20 A tekniğin dezavantajı. mikromanipülatörler kullanarak gerektirir. Mikromanipülatörler ile tanıdık Araştırmacılar, bu tekniğin çok sezgisel bulacaksınız; Ancak, böyle bir deneyime sahip bireyler gerekli ince hareketleri ile rahat olmak gerekir. Herhangi bir büyütme tekniği doğasında boyutu ve nükleotid kompozisyon dayalı belirli transkript tercihli büyütme. 4,6,7 Polimeraz zincir reaksiyonu (PCR), ters transkripsiyon polimeraz zincir reaksiyonu (RT-PCR) ve cDNA hızlı amplifikasyon temelli teknikler transkript üstel amplifikasyon uçları (YARIŞ) Sonuç olarak, lineer amplifikasyon ise Arna amplifikasyon işlemi sonuçları. Böylece, Arna prosedürünün en önemli avantajlarından biri mRNA transkript bağıl bollukları sadece doğrusal katlanarak yükseltme karşı kuvvetlenme sürecinde meydana gelen herhangi bir hata ya da önyargıları büyüterek daha iyi korumak için onun yeteneği yatıyor hataları ve önyargıları söyledi. Mikroarray analizi ile birleştiğinde Arna prosedürü benzer ya da farklı morfoloji, tedavi edilen veya tedavi edilmemiş tek hücreleri arasındaki mRNA bollukları karşılaştırma için izin verir. Ayrıca, yeni nesil sıralama için amplifiye malzeme teslim edilebilir. 5,8, prosedür için rastgele primerler kullanımı aksine, oligo-dT astarlanmalıdır prosedür önyargıları amplifikasyon yukarıda açıklanan beri Ancak, bu tür analizlerde dikkat edilmelidir 3 &#39;mRNA ve genellikle her turda daha sonra güçlendirilmiş malzemeden hafif kısalma sona erer. Bakım mikroarray sonuçları analiz bazı yanlış yok çağrılar biraz kısaltılmış güçlendirilmiş malzemeden ortaya çıkabilir gibi standart yöntemleri ile alınmalıdır. Buna ek olarak, sıralama ise sonuç gerçekten de tam 5 sağlayacaktır &#39;en orijinal mRNA sıra, bazı mRNA 5&#39; dizileri kaçırmış olabilir. Bu nedenlerden dolayı, ilâvelerle, mermi sayısı sınırlı olmalı.

A correction was made to <a href="http://http://www.jove.com/video/2634/transcriptome-analysis-of-single-cells">Transcriptome Analysis of Single Cells</a>. There were errors in the volumes used in the aRNA Amplification section.

Erratum: Transcriptome Analysis of Single Cells

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spiegelgas coverslips</td>
<td>&nbsp;</td>
<td>Carolina Biological</td>
<td>63-3029</td>
<td>&nbsp;</td>
<tr>
<td>Nunc 35x10 mm culture dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-90</td>
<td>&nbsp;</td>
<tr>
<td>Water for cell culture</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-724Q</td>
<td>For making solutions used for cell culture and rinsing coverslips</td>
</tr>
<tr>
<td>Poly-D-Lysine MW70-150K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>6407</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, ultrapure</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354239</td>
<td>&nbsp;</td>
<tr>
<td>Boric acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0252</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and glutamax</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41090-101</td>
<td>For plating cells</td>
</tr>
<tr>
<td>D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8769</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16050</td>
<td>&nbsp;</td>
<tr>
<td>Cytosine beta-D-arabinofuranoside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1768</td>
<td>&nbsp;</td>
<tr>
<td>MEM with Earle&#x2019;s salts and L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095-098</td>
<td>For growing cells</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>B27 serum-free supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillary tubes (1.5mm O.D, 100mm length)</td>
<td>&nbsp;</td>
<td>Kimble Chase</td>
<td>34500 99</td>
<td>Any borosilicate glass pipette will work as long as the proper bore size is attained. </td>
</tr>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175</td>
<td>Any solution will work as long as the components won&#x2019;t interfere with any future processing (i.e. no Ca2+ or Mg2+)</td>
</tr>
<tr>
<td>1ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309628</td>
<td>&nbsp;</td>
<tr>
<td>Needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>Gauge depends on the diameter of the pipettes</td>
</tr>
<tr>
<td>dNTP mix</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>28-4065-51</td>
<td>&nbsp;</td>
<tr>
<td>dt-T7 oligo</td>
<td>&nbsp;</td>
<td>Custom</td>
<td>Midland Certified</td>
<td>&nbsp;</td>
<tr>
<td>Second strand buffer (5x)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Y01129</td>
<td>&nbsp;</td>
<tr>
<td>DTT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Supplied with second strand buffer</td>
</tr>
<tr>
<td>E.coli DNA Ligase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100002324</td>
<td>&nbsp;</td>
<tr>
<td>DNA polymerase I</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004926</td>
<td>&nbsp;</td>
<tr>
<td>Rnase H</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18021-071</td>
<td>&nbsp;</td>
<tr>
<td>Megascript T7 kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1334</td>
<td>&nbsp;</td>
<tr>
<td>Random primers</td>
<td>&nbsp;</td>
<td>BMB</td>
<td>11034731001</td>
<td>&nbsp;</td>
<tr>
<td>Superscript III Reverse Transcriptase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>56575</td>
<td>in kit (18080-044) comes with first strand buffer (Y02321) and DTT</td>
</tr>
<tr>
<td>MEGAclear Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>1908</td>
<td>&nbsp;</td>
<tr>
<td>MinElute Reaction Cleanup Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28206</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>100004994</td>
<td>&nbsp;</td>
<tr>
<td>Rnasin</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>N251B</td>
<td>&nbsp;</td>
<tr>
<td>Illumina TotalPrep RNA Amplification Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AMIL1791</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-87</td>
<td>Sutter has many other models, many are discussed in the cookbook</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Many other micromanipulators will work such as the newer Eppendorf models</td>
</tr>
<tr>
<td>Pipette Holder</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>MP-S15A</td>
<td>Will vary with micromanipulator and pipette O.D.</td>
</tr>
<tr>
<td>Bioanalyzer RNA 6000 Nano Kit</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>5067-1511</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transcriptome analysis of single cells

Birçok gen ekspresyon analizi teknikleri heterojen toplumlarda kültür doku homojenatlarında veya hücreleri hücreleri izole malzeme güveniyor. 1,2,3 durumda beynin hipokampus gibi bölgelerin her biri, farklı hücre türleri ile karmaşık bir düzen içeren farklı mRNA profilleri. Hücre popülasyonlarının arasında ve içinde moleküler farklılıklar içine derinlemesine bir soruşturma daha fazlası için tek hücre hasat yeteneği sağlar. Biz daha fazla işlem için hücre hasat için basit ve hızlı bir yöntem açıklanmaktadır. Genellikle elektrofizyoloji kullanılan Pipetler (aspirasyon kullanarak) bir ilgi hücre izole etmek için kullanılan ve moleküler biyoloji teknikleri herhangi bir sayı ile daha fazla işlem için elverişli bir Eppendorf tüp içine depozito. Bizim protokol dendritler hücre kültürü veya akut dilim bile tek tek hücrelerin hasat için modifiye edilebilir. Ayrıca tek bir hücre izolasyonların büyük bir alt uygulama olarak Arna amplifikasyon yöntemi açıklanmaktadır. Bu yöntem, ters transkripsiyon veya gerçek zamanlı polimeraz zincir reaksiyonu (PCR) gibi diğer gen ekspresyon analizi teknikleri, alternatif olarak laboratuar tarafından geliştirilmiştir. 4,5,6,7,8 Bu teknik, doğrusal amplifikasyonu için sağlar polyadenylated RNA sadece malzeme femtograms ile başlayan ve antisens RNA mikrogram miktarlarda. Doğrusal amplifiye malzeme izole hücre transcriptome bileşenleri göreceli bolluk PCR üstel amplifikasyon daha doğru tahmin sağlar. Temel prosedür amplifikasyon iki tur oluşur. Kısaca, bir T7 RNA polimeraz promoter mRNA transkript oluşturulan çift zincirli cDNA içine dahil edilmiştir. Bir in vitro transkripsiyon geceleme (IVT) reaksiyon sonra T7 RNA polimeraz çift telli cDNA birçok antisens transkript üretir yapılır. Ikinci turda bu süreci tekrarlıyor ancak başlangıç ​​materyali bu yana bazı teknik farklılıklar ile antisense RNA. Amplifikasyon üç turda, ikinci turda tekrar standarttır. Genellikle, üretilen antisens RNA melezleşmiştir ve bir mikroarray tespit böylece biotinlenmiş nükleozid trifosfatlar kullanarak in vitro transkripsiyon reaksiyonu üçüncü turda yapılır. 7,8

Watch this Scientific Journal Video about Transcriptome Analysis of Single Cells at JoVE.com

Transcriptome Analysis of Single Cells

Bu yazıda, sıçan primer nöronal kültürlerin ve sonraki transcriptome analizi Arna amplifikasyon kullanarak tek hücre hasat için basit bir yöntem açıklanmaktadır. Bu yaklaşım, herhangi bir hücre tipi için genellenememektedir.

Tek Hücre Transcriptome Analizi

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Research

JoVE Journal

Neuroscience

29.6K Görüntüleme.  University of Pennsylvania. Bu yazıda, sıçan primer nöronal kültürlerin ve sonraki transcriptome analizi Arna amplifikasyon kullanarak tek hücre hasat için basit bir yöntem açıklanmaktadır. Bu yaklaşım, herhangi bir hücre tipi için genellenememektedir.

Video: Transcriptome Analysis of Single Cells

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Bozulmamış Duyu Organ Öncü Hücreler Canlı hücre Görüntüleme Drosophila Pupa

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Nörobilim

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sıralı Nöromusküler Kavşağı&#39;nda Tek Schwann Hücreleri belirginleştiren Photo-beyazlatma

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Merkez Sinir Sistemi Tek Hücreler Etiketleme<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Travmatik Beyin Hasarı Sonrası Nöronlar veya gen ekspresyon analizi için Tek Nöron zenginleştirilmiştir Populasyonlarının Lazer Yakalama Mikrodisseksiyon

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

Atomik Kuvvet Mikroskobu ile Beyin Polisomlar de Peering

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

Retinal Ganglion Hücrelerinin Tanımlanmış Alt Kümelerinin Tek Hücreli RNA-Seqi

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Bağışıklık hücreleri tek siyatik sinirleri ve Dorsal kök gangliyon üzerinden tek bir fare kullanarak Akış Sitometresi Analizi

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Yetişkin omurilik çekirdeği yalıtım kitlesel paralel tek-çekirdek RNA sıralama için

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

Mikroenjeksiyon kullanarak ınsan hela hücrelerinde Spliceosomal snRNA yerelleştirme Analizi

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.