Yazarlar Kas için bir standart ve kontrol pozitiflerin tür hediye için Hastalık Kontrol ve Önleme Ulusal calicivirus Laboratuvarı Merkezi (CDC) teşekkür ve reaktiflerin sağlanması için Johns Hopkins Halk Sağlığı Okulu Laboratuvarları olacaktır.

1. Tabure Örnekleri <ol><li> Dışkı örnekleri RNA korumak için dondurularak saklanmalıdır. % 10 fekal süspansiyon yapmak için, çözülmüş dışkı örneği yaklaşık 0.1 gr almak ve PBS ile 1 ml&#39;ye tamamlayın. </li><li> Önlemek için 200 ul olarak tablet dondurma ve çözme de ekledi. -70 ° C&#39;de alikots depolamak </li><li> Çözülme ve ekstraksiyon kullanılmadan önce 10 dakika süreyle 4000 g alikotları santrifüjlenir. </li></ol> 2. Guanidin &amp; Silika ile Ekstraksiyon için Silika Parçacıklar hazırlanması <ol><li> RT anda, silikon dioksit, 30 gram askıya alma ve 250 mL dH 2 O ile tamamlayın. </li><li> Sedimantasyon 24 saat sonra emme ile süpernatantı 215 ml kaldırın. 250 mL dH 2 O ekleyin ve sallayarak silis pelet askıya. </li><li> Başka bir 24 saat sonra, emme süpernatan kaldırmak ve pH 2.0 &#39;a silika süspansiyonun pH&#39;ı ayarlamak, hidroklorik asit (HCI) kullanılarak. Cam bot kısım silika süspansiyonSu Isıtıcılar ve otoklav. </li></ol> 3. Guanidin &amp; Silika ile Ekstraksiyon için L6 Tampon Hazırlanması <ol><li> Oda sıcaklığında, 0.1 M Tris hidroklorür, pH 6,4, ve bir 0.2 M EDTA, pH 8.0 11 mL çözelti 50 mL X-100 guadinyum tiyosiyanat, 60 g (GuSCN) ve Triton 1.3 g çözülerek L6 tampon hazırlanır. </li></ol> 4. Guanidin &amp; Silika ile Ekstraksiyon için L2 Tampon Hazırlanması <ol><li> Oda sıcaklığında, 0.1 M Tris hidroklorür, pH 6.4 içinde, 150 mL guadinyum tiyosiyanat ile 180 g (GuSCN) eriterek L2 tampon hazırlanır. </li></ol> 5.. Ekstraksiyon Prosedürü <ol><li> Her iki ekstraksiyon bir Kas pozitif dışkı örneği, pozitif kontrol ve DEPC işlenmiş su olarak, negatif kontrol olarak dahil edilmelidir. </li><li> Bir mikro-santrifüj tüpünde aşağıdaki karışımı; tampon L6, silika parçacıkların 20 uL 1 mL, ve% 10 dışkı ekstre süspansiyonun 200 uL ekleyebilir. Vortex kısa ve 15 mi RT de bırakınn. </li><li> 10 sn için 6000 g&#39;de santrifüje süspansiyonu ve% 70 etanol ile iki yıkama ve aseton ile bir kez ve ardından, iki kez 1 ml tampon L2 ile pelet yıkanır. 5 dakika süreyle 56 bir kuru ısıtma blok ° C pelet kurutun. </li><li> RNase içermeyen dH 2 0 ile 50 uL ilave edilerek silika pelet içinde hidrat RNA. 15 dakika boyunca RNasin 1 uL, 56 azından ters tüpü ve inkübe tarafından karışımı ° C ekleyin. </li><li> , Bir tezgah üstü santrifüj içinde, tam hızda 3 dk için santrifüje ve RNA içeren süpernatan 40 uL toplayabilir. </li><li> Nanodrop 2000 spektrofotometre (Termo bilimsel) kullanarak ayıklanan RNA kantifiye. Daha sonra -70 ° C&#39;de saklayın kullanımı kadar, 6 aya kadar. (Not: bozulmamış toplam RNA korumak için daha iyi bir yolu cDNA dönüştürerek edilir). </li></ol> 6. Ticari RNA QIAamp viral RNA Kiti Kullanarak Alternatif Ekstraksiyon Tam talimatları kitapçık Prov bulunurQIAGEN kiti ile ided. Aşağıdaki gibi kısaca prosedür: <ol><li> Tampon AVL pipetleyin 560 uL 1.5 ml tüp içine taşıyıcı RNA içeren ve% 10 dışkı süspansiyonu 140 uL ekleyin. 15 sn için darbe vorteksleme karıştırın. 10 dakika için oda sıcaklığında inkübe edilir. </li><li> 15 sn için darbe vorteksleme tarafından örnek ve karıştırın etanol 560 L (96-100%) ekleyin, ardından kısa bir süre santrifüj. </li><li> Bir 2 mL toplama tüpü içine yerleştirilmiş bir dönme kolonuna Bu çözeltinin 630 uL geçerlidir. 6,000 gr az 1 dakika santrifüjleyin, sonra temiz bir 2 ml tüp içine spin kolon yerleştirin. </li><li> 1 dakika için 6,000 g Tampon AW1 ve santrifüj 500 uL ile bağlı RNA içeren sütunun yıkayın. Temiz 2 mL toplama tüpü yerleştirin sütun. </li><li> 3 dakika boyunca tam hızda (20.000 g veya 14.000 rpm) 500 Tampon AW2 arasında uL ve santrifüj ile sütun yıkayın. </li><li> Temiz bir 1.5 mL mikro-santrifüj tüpüne koyun spin kolon, 40 uL DEPC-işlenmiş su ekleyin ve tam hızda RNA Zehir. TekrarlayınYıkanan RNA son 80 uL için ce. </li><li> Nanodrop 2000 spektrofotometre (Termo bilimsel) kullanarak ayıklanan RNA kantifiye. Daha sonra -70 ° C&#39;de saklayın kullanımı kadar, 6 aya kadar. (Not: bozulmamış toplam RNA korumak için daha iyi bir yolu cDNA dönüştürerek edilir). </li></ol> 7. Bir adım Real Time Özgül Taqman problar ile RT-PCR ile Çıkarılan RNA Norovirüs tespiti Enstrüman StepOnePlus Real Time PCR Sistemleri (Applied Biosystems). Metodoloji <ol><li> Wipe ve herhangi bir potansiyel RNaz kontaminasyonu gidermek için MESAFEDE RNaz tüm çalışma yüzeyleri, pipetler ve santrifüjler temizleyin. </li><li> Pipetleyin Kas GI ve GII tarama ana Tablo 1&#39;e göre karıştırır. Astarlar ve TaqMan probları Tablo 2&#39;de listelenmiştir. </li><li> Her reaksiyon tüpüne uygun ana karışımı kısım 10 uL. </li><li> Seyreltilmemiş bilinmeyen örneğin 5 uL ekleKarşılık gelen reaksiyon kuyucuklara sırasıyla RNA, DEPC işlenmiş-negatif kontrol olarak su, ya da gastrointestinal GII pozitif kontrol RNA. Tüm örnekleri çoğaltmak çalışması gerekiyor. Sırasıyla 300 mikrogram / uL ve 500 mikrogram / uL konsantrasyonu ile pozitif kontrol ve standartlara Hastalık Kontrol ve Önleme Ulusal calicivirus Laboratuvarı Merkezi (CDC) tarafından sağlandı. </li><li> 10 sn için 6000 g reaksiyon plaka santrifüjleyin. </li><li> Deney özellikleri ekranında; çalıştırmak için deneme türü tanımlamak ve seçin. TaqMan reaktifler reaktifler tipi ve bunun Ön-PCR okuma ve amplifikasyon seçili olarak görüntülenir emin olun. </li><li> Tablo 3&#39;te termal profilini kullanılarak 15 uL reaksiyonları çalıştırın. </li><li> Sonuçları çözümleyin. </li></ol> 8. Konvansiyonel RT-PCR ve Dizileme kullanarak Kas GI ve GII Genotiplendirme (Bir ortak ve yaygın olarak kullanılan yöntem var çünkü bu video belirtilen değil.) Çalgı. Biyosistem StepOne ve Quantitec şablonu ile StepOnePlus Real Time PCR Sistemleri uygulanır. Metodoloji <ol><li> Genotipleme önce, numunelerin genogroup (GI ya GII) RT-PCR ile yukarıda anlatılan tarama ile tespit edilir. GI Kas RNA GII genotipleme ana karışımı ile GI genotipleme master miks ve GII Kas RNA ile amplifiye edilmelidir. </li><li> Pipetleyin Kas GI ve GII genotipleme ana Tablo 4&#39;e göre karıştırır. Astarlar GI SKF / SKR ve G2 SKF / SKR Tablo 5&#39;te listelenmiştir. </li><li> 0.2 ml reaksiyon tüpleri için uygun ana karışımı kısım 45 uL. </li><li> Her bir negatif kontrol tüpüne DEPC işlenmiş su-5 uL, ve ön-elenmiş, Ara (GI ve / veya GII) karşılık gelen reaksiyon tüpüne pozitif RNA&#39;nın 5 uL ekleyin. </li><li> Tablo 6 Isı profili kullanılarak 50 uL reaksiyonları çalıştırın. </li><li> PCR ürünleri contai gönderen az 100 amplifiye kapsid bölgenin uL ng / arınma ve sıralama için Şirket Macrogen için ning. (Sekans başına 9700 Büyük Seneca Hwy. Rockville, MD 20850 ve örnek başına 10 $). </li></ol> 9. Temsilcisi Sonuçlar <img src="/files/ftp_upload/3232/3232fig1.jpg" alt="Şekil 1" /> Şekil 1 Taqman ishalli çocukların gaita örnekleri çıkarılan RNA tayini için kullanılan One-Step RT-PCR temsili sonuçlarını gösterir. Olumlu bir örnek için eşik (Ct) GII için GI ve Ct 39 daha az veya CT 37 eşit kabul edildi. Pozitif kontrol için Ct-değerleri 27 ve sırasıyla GI ve GII için ORF1-ORF2 bileşkenin 18 daha az bulunmuştur. <a href="http://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank">büyük rakam görmek için buraya tıklayın</a> . <img src="/files/ftp_upload/3232/3232fig2.jpg" alt="Şekil 2" /> Şekil 2 </strong&gt; silika yöntemi guanidinyum ve QiAmp viral RNA kit Ct değerlerinin bir head-to-head karşılaştırma gösterir. Her iki test verileri eşitlik çizgi (eğim = 1 ve önünü = 0) çok yakın düşer. Bu Regresyon Analizi ve Korelasyon Katsayısı ile teyit edilir. Her ikisi de negatif kontrol yöntemlerle değerlendirilir ve 0 olduğu bulunmuştur. <table border="1"><tbody><tr><td> Master Mix </td><td> Final Conc </td><td> Hacim (uL) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 2.875 </td></tr><tr><td> Quantitect RT-PCR Master Mix </td><td> 1x </td><td> 6.250 </td></tr><tr><td> RT Mix </td><td> 1x </td><td> 0.125 </td></tr><tr><td> 50 uM Cog R astar </td><td> 1 uM </td><td> 0.250 </td></tr><tr><td> 50 uM Cog F astar </td><td> 1uM </td><td> 0.250 </td></tr><tr><td> 10 uM Yüzük prob </td><td> 1 uM </td><td> 0.125/0.250 * </td></tr><tr><td></td><td></td><td> 10.00 </td></tr></tbody></table> Her bir prob 0,125 uL GII testleri için kullanılan süre her bir prob * 0,250 uL, GI test için kullanılmıştır. Tarama GI ve GII için Tablo 1. Qiagen Quantitect master miks (Trujillo ve ark., 2006). 7 <table border="1"><tbody><tr><td> Isim </td><td> Geno grup </td><td> Kullanmak </td><td> Dizisi (5 &#39;den 3&#39;) </td></tr><tr><td> Cog 1R </td><td> GI </td><td> Astar boya </td><td> CTT AGA CGC CAT CAT TYA C </td></tr><tr><td> Cog 1F </td><td> GI </td><td> Astar boya </td><td> CGY TGG ATG CGN TTY CAT GA </td></tr><tr><td> Cog 2R </td><td> GII </td><td> Astar boya </td><td> TCG ACG CCA TCT TCA TTC ACA </td></tr><tr><td> Cog 2F </td><td> GII </td><td> Astar boya </td><td> ARAÇ GAR BCN ATG TTY AGR TGG ATG AG </td></tr><tr><td> Halka 1A </td><td> GI </td><td> Sonda </td><td> FAM-AGA TYG CGA TCY SKK GTC CA-BHQ-1 </td></tr><tr><td> Halka 1B </td><td> GI </td><td> Sonda </td><td> FAM-AGA TCG CGG TCT SKK GTC CA-BHQ-1 </td></tr><tr><td> Ring2-TP </td><td> GII </td><td> Sonda </td><td> FAM-TGG GAG GGC GAT CGC AAT CT-BHQ-1 </td></tr></tbody></table> Tablo 2. Primer ve genogroups I, II (Kageyama ve ark. Için gerçek zamanlı kantitatif RT-PCR için kullanılmıştır prob oligonükleotidler,2003). 8 <table border="1"><tbody><tr><td> Adım </td><td> Sıcaklık (° C) </td><td> Süresi (dk) </td><td></td></tr><tr><td> 1 </td><td> 50 </td><td> 30:00 </td><td> cDNA sentezi </td></tr><tr><td> 2 </td><td> 95 </td><td> 15:00 </td><td> HotStart Taq polimerazı aktive </td></tr><tr><td> 3 </td><td> 95 </td><td> 00:15 </td><td></td></tr><tr><td></td><td> 60 </td><td> 01:00 </td><td> 45X döngüleri </td></tr></tbody></table> Tablo3. Için termal profilini Tek aşamalı Taqman Gerçek zamanlı RT-PCR (Trujillo ve ark., 2006). 7 <table border="1"><tbody><tr><td> Master Mix </td><td> Final Conc </td><td> Hacim (uL) </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 29.50 </td></tr><tr><td> 5X Qiagen RT-PCR Tamponu </td><td> 1x </td><td> 10.00 </td></tr><tr><td> 10 mM dNTP Karışım </td><td> 0.4 mM </td><td> 2.00 </td></tr><tr><td> Enzim Mix </td><td></td><td> 2.00 </td></tr><tr><td> 40 U / uL RNAsin </td><td> 20 U / uL </td><td> 0.50 </td></tr><tr><td> 10 uM SKF </td><td> 0.1 uM </td><td> 0.50 </td></tr><tr><td> 10 uM SKR </td><td> 0.1 uM </td><td> 0.50 </td></tr></tbody><tbody><tr><td></td><td></td><td> 45.00 </td></tr></tbody></table> Tablo 4. Qiagen genotipleme GI ve GII için Tek Adım RT-PCR master miks. <table border="1"><tbody><tr><td> Isim </td><td> Geno grup </td><td> Kullanmak </td><td> Dizisi (5 &#39;den 3&#39;) </td></tr><tr><td> G1SKF </td><td> GI </td><td> Astar boya </td><td> 5&#39;-CTG CCC GAA TTY GTA AAT GA - 3 </td></tr><tr><td> G1SKR </td><td> GI </td><td> Astar boya </td><td> 5&#39;-CCA ACC ARAÇ CCA TTR TAC A - &#39;3 </td></tr><tr><td> G2SKF </td><td> GII </td><td> Astar boya </td><td> 5&#39;-CNT GGG AGG KHK ATC DHA A - 3 </td></tr><tr><td> G2SKR</td><td> GII </td><td> Astar boya </td><td> 5&#39;-CCR CCN DHA TRH CCR TTR TA CAT-3 </td></tr></tbody></table> Tablo 5. Primerler kapsid Bölge Bölge C (Kojima ve ark., 2002) ve amplifikasyonu için kullanılır. 5 <table border="1"><tbody><tr><td> Adım </td><td> Sıcaklık (° C) </td><td> Süresi (dk) </td><td></td></tr><tr><td> 1 </td><td> 60 </td><td> 30:00 </td><td> cDNA sentezi </td></tr><tr><td> 2 </td><td> 96 </td><td> 15:00 </td><td> HotStart Taq polimerazı aktive </td></tr><tr><td> 3 </td><td> 94 </td><td> 00:030 </td><td></td></tr><tr><td></td><td> 52 </td><td> 01:00 </td><td> 40X döngüleri </td></tr><tr><td></td><td> 72 </td><td> 00:30 </td><td></td></tr><tr><td> 4 </td><td> 72 </td><td> 10:00 </td><td> Tavlama </td></tr></tbody></table> Tablo 6. Taqman Tek Adım RT-PCR için termal profili.

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Çıkar çatışması ilan etti.

Dışkı örneklerinden nükleik asit izolasyonu için yöntem içi ekonomik kullanarak, Qiagen gelen ticari QIAmp viral RNA kiti ile eşit sonuçlar elde ve birlikte TaqMan RT-PCR Laboratuvarımızda geliştirilen ile biz Kas geniş bir algılayabilir GI ve GII genogroup ait genotipler. Bölgenin C protokolünün yeni bir yayın% 78 6 genotipleme oranları bildirdi. Çağdaş Kas suşların çeşitliliğin önceki yıllarda artan bu yana, başarı oranı test edilen numuneler için bölgede C primerlerin uyu...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Guanidin izotiyosiyanat </td><td> Sigma-Adrich </td><td> G9277 </td><td></td></tr><tr><td> Tris HCI </td><td> Sigma-Aldrich </td><td> T5941 </td><td></td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E5134 </td><td></td></tr><tr><td> Silis </td><td> Sigma-Aldrich </td><td> S5631 </td><td></td></tr><tr><td> Triton X-100 </td><td> BDH Kimyasallar </td><td> 14530 </td><td></td></tr><tr><td> Diethylpyrocarbonate </td><td> Sigma-Aldrich </td><td> D-5758 </td><td></td></tr><tr><td> Viral RNA Mini Kit (250) QIAamp </td><td> QIAGEN </td><td> 52906 </td><td></td></tr><tr><td> QuantiTec Probe RT-PCR kiti (200) </td><td> QIAGEN </td><td> 204443 </td><td></td></tr><tr><td> Qiagen Bir Adım RT-PCR kiti (200) </td><td> QIAGEN </td><td> 210212 </td><td></td></tr><tr><td> RNaz İnhibitör 2000 adet </td><td> A.Biosystems </td><td> N808-0119 </td><td> 2000 unids / flakon </td></tr><tr><td> Non-Stick Rnae serbest mikrofuge&#39;de Tüpler </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> Ultra Saf Agaroz 1000 </td><td> Invitrogen </td><td> 16550-100 </td><td></td></tr></tbody></table>

detection and genogrouping of noroviruses from children's stools by taqman one-step rt-pcr

Noroviruses (NoVs) her yaştaki insanlarda dünya çapında sporadik akut gastroenterit salgınlarının önde gelen nedenidir. Onlar Rotavirüs benzer bir halk sağlığı etkisi olan çocuklarda hastaneye yatış önemli nedenlerindendir. NoVs büyük genetik çeşitlilik RNA virüsleri vardır ve yeni suşlar sürekli bir görünümü vardır. Beş genogroups tanınır; GI ve GII onların birçok genotip ve insan enfeksiyonu için en önemli olan alt tipleri ile. Bununla birlikte, bu iki genotiplerinin tan teşhis ve tedavi geciktirilmesi, bir problem teşkil etmektedir. 1, 2, 3 Dışkı örneklerinden RNA ekstraksiyonu için en sık kullanılan yöntem Qiagen gelen QIAmp viral RNA ticari kiti. Bu yöntem, tamponlar, bir silika jel membran bağlanma özelliklerini birleştiren bir kontrol RNases ve microspin hızı ile birlikte kolonuna RNA bağlama optimum sağlar. Bu yöntem basit, hızlı ve güvenilir ve Carr iseüretici tarafından sağlanan açıklama ayrıntılı olarak bir kaç adımda ied. Norovirüs ishalin en sık nedeni olarak rotavirüs sadece ikinci. Norovirüs tanı ishal patogenezinde tüm çalışmaların yanı sıra salgınlar ya da bireysel ishal vakası mevcut olmalıdır. Şu anda ancak norovirüs tanı tanı basit bir yöntem olmaması nedeniyle sadece birkaç merkezde sınırlıdır. Bu gecikme teşhis ve tedavi 1, 2, 3. Buna ek olarak, ülke içinde ve arasındaki maliyet ve korozif tamponların düzenlenmiş ulaşım nedeniyle lojistik problemler ortaya çıkarır, bu mamul kitleri kullanın. Sonuç olarak, bu protokolde, orijinal Bom dayalı bir alternatif, ekonomik, in-house yöntemi tarif ark guadinyum tiyosiyanat ile anti-nükleaz özellikleri ile birlikte silika partiküllerinin nükleik asit bağlama özellikleri kullanır. Yöntemi 4 . <p class=Dışkı örnekleri, farklı hedefler kullanıldığı birkaç RT-PCR protokollerden NoVs izolatlarının tespiti ve genogrouping (GI ve GII) için &quot;jove_content&quot;&gt; geliştirilmiştir. Uzlaşma yüksek duyarlılık, özgüllük ve yüksek verimlilik ve kullanım kolaylığı ile tekrarlanabilirliği birleştirir çünkü TaqMan kimya kullanarak bir RT-PCR, tanı için en iyi moleküler teknik olacağını olmasıdır. Burada, açık okuma çerçevesinin 1 (ORF1)-ORF2 bileşke bölgeye yönelik bir deneyde tarif; teşhisi için böylece en korunmuş Ara genomunun bölgesi ve en çok uygundur. Daha da gen analizinde kapsid (Bölge C) aslen Kojima ve arkadaşları tarafından tarif primerler kullanılarak. 5 büyük protein son derece değişken N-terminal-kabuk hedef geleneksel bir RT-PCR için ayrıntılı olarak verilmiştir. Konvansiyonel PCR ile PCR ürün Sekanslayan GI ve GII genogroups ait genotiplerinin farklılaşma sağlar.

Watch this Scientific Journal Video about Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR at JoVE.com

Detection and Genogrouping of Noroviruses from Children&#039;s Stools By Taqman One-step RT-PCR

Astar ve açık okuma çerçevesi 1 (ORF1)-ORF2 kavşak bölgesidir Norovirüs genomunun en korunmuş bölgeye özgü TaqMan probları kullanan çocuk tabureleri, gelen algılama ve Norovirüs izolatların genogroup belirlenmesi için Tek Adım RT-PCR nitelendirdi. Bir ticari olmayan, maliyet-etkin RNA ekstraksiyon yöntemi ayrıntılı olarak verilmiştir.

Taqman By Çocuk Tabureler gelen Noroviruses tespiti ve Genogrouping Tek aşamalı RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

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Research

JoVE Journal

Medicine

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

17.1K Görüntüleme.  Universidad Peruana Cayetano Heredia. Astar ve açık okuma çerçevesi 1 (ORF1)-ORF2 kavşak bölgesidir Norovirüs genomunun en korunmuş bölgeye özgü TaqMan probları kullanan çocuk tabureleri, gelen algılama ve Norovirüs izolatların genogroup belirlenmesi için Tek Adım RT-PCR nitelendirdi. Bir ticari olmayan, maliyet-etkin RNA ekstraksiyon yöntemi ayrıntılı olarak verilmiştir.

Video: Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Kantitatif Gerçek-zamanlı PCR (qPCR) tarafından Prostat tümörlerinde mikroRNA Algılama

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Tıp

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Kanser Tedavisi İzleme Cerenkov Lüminesans Görüntüleme (CLI)

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Fare Modeli Inferior Vena Cava İnterpozisyon Greft İmplantasyonu

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Hastalık Modelleme için kas biyopsisi Hasta türetilmiş Hücre Hatları İzolasyonu ve Ölümsüzleştirme

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Tüm Doku ve Biyopsi İnsan Epitelyal Enteroids ve Colonoids kurulması

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

Pankreas Kanalı infüzyonu: İlaç ve Viral Doğumda Etkili ve Seçici Bir Yöntem

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Birincil fare tetkikine yalıtım doğmakta olan Protein sentez analiz radyoaktif L-azidohomoalanine etiketleme yöntemi tarafından için

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Metalik yabancı cisim yeme çocuklar için el Metal detektörü tarama

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Nitrik oksit VASP fosforilasyon ölçme tarafından kan trombosit tabanlı algılama

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

Farelerde hyaloid damarlarının değerlendirilmesi ve karakterize edilmesi

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...