This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).

endoskopik retrograd kolanjiopankreatografi ile hastaların safra elde edilmesi (ERCP) Kurumsal Değerlendirme Kurulu tarafından bir insan denekler çalışma protokolünün onaylanması gerekiyor. Burada sunulan tüm çalışmalar Johns Hopkins Üniversitesi Kurumsal Değerlendirme Kurulu tarafından onaylandı. Bile 1. Exosome İzolasyon <ol><li> endoskopik retrograd kolanjiyopankreatografi (ERCP) gerçekleştirin. yatar pozisyonda hastayı ve entübe. <ol><li> Hastanın ağız yoluyla bir duodenoskop Pass ve duodenum ikinci bölümü takın ve büyük papilla belirlemek. </li><li> Seçici bir üçlü lümen 0.035 inç (0.9 mm) hidrofil kılavuz tel ile önceden yüklenmiş sfinkterotom takarak koledok cannulate. </li><li> vesileyle sağ hepatik kanalından kistik kanal ayırt etmek zor olabilir çünkü sol hepatik kanala floroskopi altında kılavuz teli ilerletmek, daha sonra ilerlemekistikrarlı bir pozisyon elde etmek için safra kanalına 5 cm sfinkterotom. </li><li> Luer kilidi sayesinde kılavuz tel bağlantı noktasına 10 ml şırınga takmak, kılavuz teli çıkarın ve negatif basınç 10 ml kullanılarak safra aspire. </li><li> Kılavuz teli reinserting ve safra yolları opaklaşmaz için enjeksiyon portu üzerinden kontrast madde enjekte edilerek ERCP ile devam etmek safra içeren şırınga çıkarın. </li><li> buz üzerinde 15 ml tüp içine toplanmış safra aktarın ve santrifüj adımla devam hazır olana kadar buz üzerinde tutmak. Dikkat: Kendinizi ve örnekleri korumak için eldiven giyin! o Hepatit A virüs partikülleri içerebilir olarak potansiyel infeksiyon kaynağı olarak safra davranın! </li><li> buz üzerinde örnek (ler) tutun. Her iki durumda da, 10 dakika boyunca 4 ° C&#39;de 500 x g&#39;de adım 1.2 veya santrifüj ile devam etmek ve daha sonra kullanılana kadar -80 ° C&#39;de dondurun. Not: birkaç yıl bu şekilde depolanmış numuneler rutin iyi sonuçlar 19 kullanılmıştır. </li></ol&gt; </li><li> mikrosantrifüj tüpü içine transferi safra 400 ul ve 4 ° C&#39;de 10 dakika boyunca 300 x g&#39;de santrifüj ile hücreler ve hücre artıkları toplar. </li><li> , Pipetleme süpernatantı taze mikrosantrifüj tüpüne transfer ve daha fazla kalıntı yüzey maddesi temizlemek için 4 ° C&#39;de 20 dakika daha 16,500 x g hızında örnek (ler) santrifüj. biohazard atık tüpleri atın. Alternatif olarak, yerine ultrasantrifüjdeki örnekleri dönerler. </li><li> Süpernatant toplayın ve sol nm 200 daha büyük herhangi bir parçacıkları çıkarmak için bağlayıcı iyi akış hızı ve düşük protein olan 0.2 mikron polyethersulfone (PES) membran filtreden bir şırınga ile bir biyogüvenlik kabini filtre. </li><li> Pipet süzülmüş süpernatant tüpleri ultrasantrifüjdeki ve tüpler fazla 2/3 tam böylece PBS ekleyin. Tüpler daha az likit içeriyorsa, tüpler santrifüj sırasında çökecek! Tüpler, yıkandı, otoklavlanmış ve birkaç kez tekrar edilebilir. </li><li> pellve 4 ° C&#39;de 70 dakika boyunca 120,000 x g&#39;de ultrasantrifugasyon ile eksozomlar. bazen küçük sarı topaklar görmek için ultrasantrifüjdeki tüpün dibinde santrifüj bakmak sonra; Bu eksozomlar bulunmaktadır. </li><li> dikkatli bir şekilde boşaltma yoluyla süpernatan atılır ve% 10 v / v nihai bir konsantrasyona kadar ağartıcı ekleyerek atık dezenfekte ve 20 dakika boyunca bekletildi. </li><li> Alt deneyler için, ya da uygun bir çözelti küçük bir hacme pelet (lar) işleme (örneğin, radyoimmunopresipitasyon analizi (RIPA) Protein izolasyonu, mıR izolasyonu için RNA liziz tamponu için tampon A) ya da fosfat 50-150 ul içinde tekrar süspansiyon tamponlu ya TEM veya nanopartikül optik analizle eksozom hazırlama karakterizasyonu için -80 ileride kullanmak üzere ° C (kantitatif gerçek zamanlı polimeraz zincir reaksiyonu (qRT-PCR) ya da protein çıkarımı vs.) depolanabilir veya kullanılabilir tuz (PBS) . </li></ol> tarafından 2. Exosome KimlikTransmisyon Elektron Mikroskobu (TEM) ya da cryoEM <ol><li> pipetleme PBS 100 ul dışı kesecikler süspanse. İlk kez izole performans özellikle İdeal olarak, -20 ° C&#39;de saklandı veya edilmemiş -80 ° C eksozomlar kullanımı. </li><li> 2 dakika boyunca 400 meş karbon kaplı Parlodion bakır ızgaralar için 20 ul bir kısım (yaklaşık olarak 2 ug) adsorbe oda sıcaklığında kurumaya bırakın. </li><li> kurutuldu hazırlanması dikkatle damla yerleştirerek oda sıcaklığında 5 dakika boyunca% 1 (h / h), glutaraldehid (EM dereceli) &#39;de eksozom düzeltildi. </li><li> 5 dakika boyunca iki kez su ile ızgaralar yıkayın ve daha sonra 30 saniye boyunca% 1 fosfotungstik asit (PTA) boyanan EVs kontrast. </li><li> 80 kilovolt bir ivme gerilimi 20,000X büyütmelerde başlayan ve parçacıkların boyutu belirlenirken 100,000X için artan bir elektron mikroskobu ile görüntü kazanır. Not: Diğer preparatlarıyla Formvar karbon kaplı 200 örgü bakır veya nikel ızgaraları ve kontrast boyama li üzerine katman gibi10-15 dakika ke% 1 veya% 2 uranil asetat da mümkündür. </li><li> (: 3 oranında 1) cryoEM için, 60 ul protein G altın 10 nm ile 20 | il bir kısım (yaklaşık olarak 2 ug) karıştırılır. Bu exosome çapını belirlemek için yardımcı olacaktır. </li><li> Glow-deşarj C-düz holey karbon ızgaraları üzerine örnekleri aktarın ve blot fazla sıvıyı çıkarın. sıvı etan içine hemen onları batırarak ızgaraları dondurun. </li><li> Sıvı azot içinde cryoholder ızgaraları monte edin. </li><li> 20,000X ve 100,000X büyütmelerde 200 kV görüntü kazanır. </li></ol> Çok parametreli Nanoparçacık Optik analizi ile 3. Exosome Kimlik <ol><li> pipetleme PBS 100 ul izole eksozomlar süspanse. </li><li> çeşitli oranlarda 600 ul PBS içinde eksozomlar sulandırınız (benzeri 1:50, 1: 100, 1: 300, 1: 600 ile 1: 1,200). </li><li> üreticilerinin göre, gerçek zamanlı olarak uygun bir araç ve yazılım ile lazer ışını tarama ile eksozomlar görselleştirmeRER protokol 20. </li><li> parçacıklar görünür hale gelene kadar sonra parçacıklar düzgün görünmesi için odağı ayarlamak, çekim modunda kameranın düzeyini artırmak. </li><li> standart modda, parçacıklar hala görülebilmektedir en düşük seviyesine kamerayı azaltmak iken gerekli kamera deklanşörü ve kazanç ayarlamak durumunda, bunu başarmak için. </li><li> Görme üniteyi temizlemek ve PBS seyreltme ayarlamak değilse, 20-100 parçacıklar ekranda görünür olduğundan emin olmak için kontrol edin. </li><li> Ardından 30-60 arası sn yakalama süresini ayarlayın. Video çekildikten sonra, tüm partiküller ekranda tanımlanır kadar işleme parametrelerini ayarlamak ve video dosyasını işlemek. </li></ol> Western Blot tarafından 4. Exosome Karakterizasyonu <ol><li> buz soğukluğunda Radyoimmunopresipitasyon tahlilinde 100 ul safra 2-5 mi den Lyse eksozom peletleri (RIPA) pipetleme bir proteaz inhibitörü ile tampon ve aşağı doğru tamamen ve 30 saniye boyunca kuvvetli bir şekilde karıştırın lizatlarıBir girdap üzerinde etkili bir proteinleri çözünür. </li><li> çoğu zaman gerekli olmasa da, tam parçalama sağlamak için 10 saniye, her 2 darbeleri ile buz üzerinde numunelerin darbe sonikasyon kullanın. </li><li> 5 dakika boyunca 4 ° C&#39;de 15,000 x g&#39;de santrifüj ile çözünmeyen malzemenin pelet ve temiz bir tüp süpernatantı aktarın. </li><li> Tercih edilen bir yöntem, üreticinin protokolüne uygun olarak (yani, bisinkoninik asit (BCA) Coomassie, modifiye edilmiş Lowry, 660 nm Protein Deneyi, vs.) protein konsantrasyonunu belirleyin. aynı yöntem kullanılır deneyde mukayese edilebilir sonuçlar elde etmek için sürekli olarak kullanıldığı gibi bir yöntem sürece önemli değildir. </li><li> elektroforezi (PAGE) bir poliakrilamit jel kuyuya 5 dakika boyunca 95 ° C &#39;de örnek ısıtılmasından sonra yükleme Laemmli tamponu içinde örnek başına proteinin 50 ug. </li><li> 1.5 saat PAGE jelleri üzerinde örnekleri Electrophorese bir naylon zara ayrılmıştır proteinleri aktarın. </li><li> Bdaha sonra bir seyreltmede anti-CD63 ve anti-TSG-101 gibi bir eksozom özgü antikorlar ile inkübe 1 saat süre ile% 0.1 Tween 20 (TBS-T) Tris-tamponlu tuzlu su içinde% 5 yağsız süt ile membran (lar) kilidi 1: 500, tercihen O / N 4 ° C&#39;de. </li><li> TBS-T 3 x 10 dk membranları yıkamak, sonra oda sıcaklığında 1-2 saat TBS-T içinde% 5 yağsız süt içinde HRP-konjüge edilmiş ikincil antikor eşleşen uygun olan inkübe edin. </li><li> membran (lar) TBS-T ile 3 x 5 dk yıkayın ve üreticinin protokolüne uygun olarak kemilüminesans reaktifleri ile spesifik proteinlerin algılar. </li><li> Resim film veya üreticinin protokolüne göre bir dijital görüntüleme sistemi ile membranlar. </li></ol> İzole eksozomlar 5. mikroRNA İzolasyon Not: Safra eksozom gelen miRNA&#39;nın izolasyonu piyasada mevcut olan kit göre bir tadil edilmiş MiRNA izolasyon kullanılarak gerçekleştirilir, ancak herhangi bir başka RNA izolasyon yöntemi kullanılabilir veya adapte edilebilir. <ol><li> Ekle 300# 181; l lizis / (400 ul safra izole) exosome pelet, girdap, pipet veya tamamen homojen bir lizat elde etmek eksozomlar lyse bir iğne / şırınga geçmek bina tampon. </li><li> miRNA Homojenat Katkılı 1/10 (30 ul) hacim ekleyin ve karıştırın veya tüp birkaç kez ters çevirerek iyice karıştırın. </li><li> Buz üzerinde 10 dakika süre ile bir karışım inkübe edin. </li><li> asit fenol hacmi ekleyin: Initial Homojenat (300 ul) ile eşit kloroform. </li><li> 30-60 saniye için 5 5 fmol / ul Cel-miR 39 ul ve vorteks ekleyin. </li><li> 10.000 RT, sulu (üst) ayırmak için en xg ve organik (alt) fazlar, 5 dakika boyunca santrifüj. </li><li> transfer hacmi belirterek ederken dikkatli yeni bir tüp pipetle ve transferi ile üst faz aspire. uygun bir organik çöp bidonuna organik faz atın. </li><li> küçük RNA iyice TU vorteks veya çevrilmesi ile geri kazanılan sulu faza 1/3 hacim% 100 etanol eklenir ve karıştırılır zenginleştirmek içinbirkaç kez. </li><li> Pipetleyin bir seferde 700 ul filtre kartuşunun üzerine lizat / etanol karışımı. </li><li> 10,000 x maksimum 15 saniye kartuşları santrifüj veya filtre ile karışımı geçmek vakum uygulayın. </li><li> süzüntü toplamak ve lizat / etanol karışımı 700 ul aşarsa, yeni bir tüp akış yoluyla aktarmak ve tüm süzüntüler toplamak için işlemi tekrarlayın. Bu filtre, küçük RNA&#39;ların yoksun RNA fraksiyonları içeren ve bu büyük RNA&#39;ların kurtarmak için kullanılabilir. </li><li> Toplam süzüntüye RT% 100 etanol 2/3 hacim ekleyin ve vorteks ile iyice karıştırın. </li><li> Pipet ikinci bir filtre kartuşu üzerine filtre ürünü / etanol karışımı. seferde uygulanabilecek maksimum hacim 700 ul gibi önce. </li><li> 10.000 x g&#39;de 15 saniye santrifüj veya adım 5.10 açıklandığı gibi vakum uygulamak. </li><li> akış oranı atın ve filtrelendi süzülen madde / etanol karışımı tüm kadar yineleyin. </li><li> 700 uygula81; filtre kartuşu l miRNA Yıkama Çözeltisi 1, 5-10 sn veya vakum uygulanır ve flow-through atmak için santrifüj. </li><li> Adım 5.16 yapıldığı gibi 500 ul yıkama solüsyonu 2/3 ile filtre yıkayın. </li><li> Adım 5.16 gibi yıkama yıkama solüsyonu 2/3 500 ul ile ikinci kez tekrarlayın flow-through atın ve geri toplama tüpünün filtreyi yerleştirin. </li><li> Filtreden kalan tüm sıvıyı almak için 10,000 x g&#39;de 1 dakika için filtre kartuşunu dönerler. </li><li> taze toplama tüpünün içine filtre kartuşunu aktarın ve filtrenin merkezine sıcak (95 ° C) nükleaz serbest su 100 ul geçerlidir. </li><li> RNA kurtarmak için maksimum hızda 20-30 saniye derlemeler Spin. </li><li> eluat toplayın ve hemen kullanın veya -80 ° C&#39;de saklayın. </li></ol>

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None of the authors have competing financial interests.

güvenilir safra izole eksozomlar yararlanmak için, karşılığında yüksek kaliteli örnekleri elde etmek için tutarlı, yüksek kaliteli izolasyon yöntemleri kullanılması önemlidir. Bu yazıda tanımlanan metodoloji, insan safra gelen eksozomlar ve miRNA izole etmek köklü bir yoldur. Bu en azından elektron mikroskobu ya da nanoparçacık optik analiz ve Western lekeleri içermelidir izole Eksozomlann karakterizasyonu birkaç önemli adımlar vurgulamaktadır. o miRNA istikrar ge...

Diğer biyolojik yani sıvıların, anne sütü, plazma veya idrar gibi, safra ekzozomlar, lipit zengin veziküller 1-4 içerir. Eksozomlar neden veya alıcı hücreler 5, 6, normal fonksiyon 4, 7-9 bir parçası olabilir, hücre-hücre iletişiminin bir biçimde biyolojik fonksiyonları değiştirebilir. Eksozomlar tanı 10 biyomarkerların değerli bir kaynak sağlayabilir miRNA türlerini içerebilir. miRNA&#39;lar profilleri hastalıkların 11-14 çeşitli tanısal ve prognostik belirteç olarak son yıllarda önemli önem kazanmıştır. miRNA kendisi biyolojik sıvılarda, muhtemelen ölü hücrelerden salınan ve döken hücrelerin miktarı büyük ölçüde altta yatan hastalığa etkilemiş olabilir bulunabilir. Eksozom miRNA profili mutlaka kaynak hücresine 6, 10, 15-17 miRNA profili yansıtmaz, henüz ekzozomlar ebeveyn ce için karakteristik olabilir miRNA imzaları taşıyabilirBir tümör hücresi gibi olacağım. Tümör kaynaklı eksozomlar, akciğer adenokarsinom, prostat kanseri ve diğer tümörlerin 8, 16, 18 olan hastaların plazmasında belirlenmiştir. Bu yöntemin amacı, onların önceki kullanım prosedürleri büyük ölçüde bağımsız insan safra örneklerinden gelen eksozomlann güvenilir ve sağlam izolasyon olduğunu. Böyle kolanjiokarsinomlar 19 olarak safra kanalının hastalıkların potansiyel tanısı için miRNA güvenilir bir kaynak olarak safra kullanmak için geliştirilmiştir. Bu eksozomlar izole etmek hızları artan santrifüj birkaç aşamadan oluşmaktadır ve diğer biyolojik sıvılarda uygulanabilir olabilir.

<table><tbody><tr><td>0.2 &amp;micro;m Polyethersulfone (PES) membrane filter</td><td>Corning</td><td>431229</td><td></td></tr><tr><td>thinwall polyallomer ultracentrifuge tubes&amp;nbsp;</td><td>Beckman Coulter</td><td>331374</td><td></td></tr><tr><td>SW40Ti rotor</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>LM10-HS&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>Nanoparticle Tracking Analysis (NTA) software&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>mirVANA</td><td>Life Technologies</td><td>AM1560</td><td></td></tr><tr><td>Complete Protease Inhibitor</td><td>Roche distributed by Sigma-Aldrich</td><td>4693116001</td><td></td></tr><tr><td>Immobilon PSQ</td><td>Millipore, Bedford MA</td><td>ISEQ00010</td><td></td></tr><tr><td>Anti-CD63 antibody</td><td>Abcam</td><td>ab59479</td><td></td></tr><tr><td>Anti-Tsg101</td><td>Abcam</td><td>ab30871</td><td></td></tr></tbody></table>

isolation and profiling of microrna-containing exosomes from human bile

son üç yılda exosome araştırma büyük ölçüde biyomarkerların ve bunların terapötik kullanımları tanımlanması ve karakterizasyonu doğru kapsamını genişletmiştir. Eksozomlar en son microRNA (MIRS) içerdiği gösterilmiştir. MIRS kendileri tanısal amaçlı değerli biyomarkerların olarak ortaya çıkmıştır. klinik ve hastanelerde numune toplama oldukça değişken olduğundan, tüm safra gelen miRNA izolasyon önemli ölçüde değişir. basit bir şekilde toplanan safra örnekleri, sağlam, güvenilir ve tekrar MiRNA profilleri elde etmek için izole etmek ve safradan eksozomlar karakterize etmek için yüksek kaliteli bir protokolünün geliştirilmesi gerekmektedir. yöntem, birkaç santrüfüj ve izole eksozomlar pelet nihai ultrasantrifügasyon adım bir filtre etme adımı gerektirir. Elektron mikroskobu, Western blot, flow sitometri ve çok parametreli nanoparçacık optik analiz, mevcut, e kalitesini doğrulamak için çok önemli karakterizasyon adımlardırxosomes. Örneğin Caenorhabditis elegans gibi bir türden gelen bir spesifik olmayan sentetik miRNA&#39;nın ile lisat spike bu eksozom Mirna izolasyonu için, Cel-miR-39, RNA ekstraksiyon verimi normalleştirilmesi için önemlidir. Bu yöntem aşağıdaki safra sıvısından eksozom izolasyonu başarılı MiRNA -80 ° C&#39;de birkaç yıl boyunca saklanabilir safra örnekleri profil sağlar.

eksozomlar normal mikroskobu ile tespit ya da akış sitometrisi için çok küçük olduğu için, elektron mikroskopisi ya da optik nanopartikül analizi gerçekleştirilmelidir. nanoparçacık optik analizi, aynı zamanda niceliksel ve boyutu dağılımına ve konsantrasyonu sağlar elektron mikroskobu üzerinde bir avantaja sahiptir. alet numune içine cam prizmadan ince odaklanmış lazer ışını tanıttı. İzole veziküllerin Brown hareketi bir EMCCD yüksek hassasiyet kamera il...

Watch this Scientific Journal Video about Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile at JoVE.com

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

İzolasyon ve profil MicroRNA içeren İnsan Bile dan ekzozomlar

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

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Research

JoVE Journal

Medicine

10.2K Görüntüleme.  Johns Hopkins University School of Medicine. Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Video: Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

Küçük RNA miktarının belirlenmesi Sigara kodlama için yüksek yoğunluklu lipoproteinler izolasyonu

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Biyokimya

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Protein sindirimi, Ultrafiltrasyon ve boyutu dışlama Kromatografi insan kan plazması ve Serum Exosomes yalıtım en iyi duruma getirmek için

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

SiRNA kanser hücrelerinin teslim için Exosomes hazırlanması

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Kanser Araştırmaları

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

İzole, sıralama ve insan Mezenkimal Kök hücreleri ekstrasellüler mikroRNA'lar analiz

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetik

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

HIV-1 pozitif Bireylerin Plazma eksozomlann izolasyonu

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

İmmünoloji ve Enfeksiyon

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA İçeren Exosomes İzolasyonu ve Karakterizasyonu

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Biyoloji

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Üzerinden Ultrafiltrasyon tekniği Santrifüjü kullanarak fare Bronchoalveolar lavaj sıvı ekstrasellüler veziküller yalıtım

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Kan ve Kültür Medya türetilen Exosomes saflaştırılması ve microRNA Profil

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Insan serumundan küçük kodlayıcı olmayan RNA&#39;lar izolasyonu

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

Yüksek Saf Yüksek Yoğunluklu Lipoprotein gelen miRNA Yüksek koymak Through-İzolasyon için hızlı ve basitleştirilmiş Yöntemi

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...