Bu eser desteklenen bir SC INBRE hibe--dan Ulusal Enstitüsü genel tıp bilim - NIH (RG P20GM103499) ve CA555536 & CA154664 P.H.H. için Ulusal Kanser Enstitüsü'nden verir Bu program kısmen araştırma kaynakları (5 P20 RR016461) Howard Hughes tıp Enstitüsü öncesi üniversite ve lisans bilim eğitim programı aracılığıyla Charleston Koleji için bir hibe ve hibe Milli Merkezi tarafından desteklenen yapıldı. ve Ulusal Genel tıbbi Bilimler Enstitüsü (8 P20 GM103499) ulusal sağlık Enstitüleri üzerinden.

1. Kültür ve metabolik etiketleme
<ol><li>Bir kez bir steril 1,5 mL mikro-santrifüj tüpleri kap ortasına doğru havalandırma izin vermek için bir 18'lik iğne ile poke. İlave steril 7 h 9 500 µL aşılamak suyu M. smegmatis 5 µL bir marş--dan ile Kültür yetişkin gece12. Not: Tarifi için bir 7H 9 litre suyu: 4,7 g ticari 7H 9 tozu, 2 mL gliserol, 1 mL Tween 80, 0,85 g NaCl, 5 g Albumin, 2 g dekstroz, (Toplam hacim 1 M'ye getirmek için) H2O.<ol>
<li>
(Dikkat) Standart radioprotection yöntemleri 20 µCi/mL [32P] Na2HPO4kültür ortamıyla spike. Bakteri bir 9 mm kalınlığında akrilik kalkanın arkasında yerleştirilen bir shaker kuluçka 37 ° C ve 1200 rpm ajitasyon, büyümek.</li>
	</ol>
</li>
	<li>Midlog aşamasında (O.D.600 nm ~ 0.5), tüm kültür 2 mL vida-Kapiler tüp içine aktarmak ve 10.000 x g., 2 dk toplamak için süpernatant içeren unincorporated radyoaktif orthophosphate içinde oda sıcaklığında radyoaktif bakteri centrifuging tarafından cips uygun atık konteyner.</li>
</ol>2. Toplam RNA'ların hazırlanması
Not: Toplam RNA'ların (guanidinium thiocyanate, fenol ve kloroform içeren) ticari bir RNA ayıklama reaktif kullanarak bakteriyel çökeltilerini ayıklanır üreticinin protokol sonrası.
<ol><li>1 mL ticari RNA ayıklama reaktif ve yaklaşık 200 µL cam boncuk Pelet üzerine ekleyin. Tüp güvenli bir şekilde kap ve bir homogenizer en fazla ajitasyon altında 2 min için bakteriyel bozabilir (beş numaralı seçilen araç üzerinde ayarlama ( Tablo reçetesigörmek)).</li>
	<li>Santrifüj örneği 12.000 x g 4 ° C'de 10 dakika ' Yeni bir 2 mL tüp süpernatant aktarım ve boncuklar uygun şekilde atın. Kloroform 0.2 mL ekleyin ve tüp el ile 12.000 x g de 15 s. santrifüj 4 ° C'de 15 dakika için shake şiddetle</li>
	<li>Sulu faz örnek yeni bir tüp içine tüp 45 ° de olta balıkçılığı toplamak. Interphase veya organik katman çizim kaçının. 2 renkli precipitant µL ve % 100 isopropanol 0.5 mL ekleyin. Tüp el ile 12.000 x g de 5 s. santrifüj 4 ° C'de 10 dakika için shake şiddetle</li>
	<li>Tüm tüpünden süpernatant kaldırmak ve uygun şekilde atın gibi sıvı kesir radyoaktivite izleri içerebilir. 5 min için Kuru Pelet hava sonra 200 µL serum sodyum sitrat (SSC), % 0,1 (w/v) Sodyum Lauryl Sülfat (SDS) son X 2 resuspend. Not: Stok 20 bileşimi X SSC 3,0 M NaCl, 0,3 M sodyum sitrat, pH 7,0 olduğunu.</li>
</ol>3. 96 iyi yazıcı plakaları hazırlanması
Not: tRNA microarrays el ile kırk iki 70-mer DNA oligonucleotides 3' sonuna bir 8-pin arrayer kullanarak (terminal CCA hariç) her M. smegmatis tRNA tamamlayıcı birlikte yazdırılır. DNA Oligonükleotid probları yanı sıra iyi plaka ve dizi sonda düzende dizilerini sağlanır (ek dosya 1 - sayfa 1-3 sırasıyla).
<ol><li>Tasarım DNA ilk alınırken tRNA dizileri veya genomik tRNA gibi tRNA veritabanlarındaki tRNA kodlama genler tarafından veritabanı13sondalar. Söve korunmuş 3' ucu kodladıysanız CCA. Tamamlayıcı elde edilen sıra ters. Sipariş sonda üzerinde ilk 70 nükleotit dayalı.</li>
	<li>Kuru DNA probları suda resuspend. Probları konsantrasyon 100 µM için ayarlayın. 96 bir şey-tabak içinde 3 X SSC, %0.01 (w/v) SDS final (seyreltik 60 µL bireysel iyi hisse senedi probları,-ile 6 X SSC'yi destek veriyor, %0.01 (w/v) SDS 60 µL. 50 µM oligos 120 µL hazırlamak</li>
	<li>Plaka folyo buharlaşma veya kontaminasyonu önlemek, hemen donma ve -20 ° C de ihtiyaç kadar tutmak için kapağı.</li>
</ol>4. dizi yazdırma
<ol><li>96-şey plaka (yaklaşık 20 dakika sürer) oda sıcaklığında erimek.</li>
	<li>Amin kaplı cam slaytlar bir elmas kalemi ile etiketleyin. Slaytlar dizin oluşturma birim içine yerleştirin. Kılavuz yerleşimler izleyin ve aşağıdaki gibi yapın.<ol>
<li>Dikkatle Wells Çoğalıcı iğne batır.</li>
		<li>Yavaşça dizi en az basınç (küçük bir direnç hissedeceksiniz) kullanarak cam slaytlarda yazdırın. Blok a ile bitene arrayer temizlemeden yazdırmaya devam etmek</li>
	</ol>
</li>
	<li>Hazır olduğunuzda sonraki bloğa geçmek, aşağıda açıklanan çamaşır yordamı izleyin.<ol>
<li>Çoğalıcı %5 çamaşır suyu daldırma ve hafifçe çalkalanır.</li>
		<li>Çoğalıcı emici kağıt basın.</li>
		<li>Çoğalıcı distile su içinde daldırma, yavaşça sallayın ve kağıt basın. Bir kez bu adımı yineleyin.</li>
		<li>Çoğalıcı isopropanol içinde daldırma, yavaşça sallayın ve kağıt basın.</li>
		<li>Çoğalıcı yaklaşık 20 için fan üzerinde kuru s 96-şey plaka sonraki bloğuna geçmeden. Parmak izi istediğiniz sayıya devam.</li>
	</ol>
</li>
	<li>Slaytların crosslink oligos 254-nm UV crosslinker kullanarak slayt için Kuru izin.<ol>
<li>Enerji seviyesi 999,990 µJ/cm2olarak ayarlayın.</li>
		<li>Crosslinker temiz bir yüzey üzerinde slaytlar yüz koy. "Başlat" tuşuna basın.</li>
	</ol>
</li>
	<li>Bir manyetik karıştırıcı, oda sıcaklığında ve yavaş ajitasyon altında çözüm engelleme 450 mL gecede dizide engelleyin. Toplamak ve dört kat engelleme çözüm yeniden. Not: Engelleme çözüm için reçetedir: 125 mM fosfat tampon (NaH2PO4/Na2HPO4-) pH 7.2, %2.25 SDS (w/v), 0,5 mM EDTA, 0.5 X SSC, % 1 BSA (w/v).</li>
	<li>500 mL distile su içinde oda sıcaklığında 2 kez 5 min için slaytlar yıkayın. Slaytlar için 10 centrifuging tarafından kuru s Mikroarray santrifüj oda sıcaklığında. Mağaza diziler için 6 aya kadar kuru ve karanlık bir yerde.</li>
</ol>5. dizi hibridizasyon
<ol><li>Hibridizasyon için önceden slaytlar kaynar suda yıkayın ve tarafından Santrifüjü (4.6) olduğu gibi kuru.</li>
	<li>Dizi içinde hibridizasyon kaset yerleştirin ve radiolabeled RNA örnek yükleyin.</li>
	<li>Otomatik hibridizasyon yıkama istasyonu maksimum tekrarlanabilirlik için çalıştırın.<ol>
<li>Program aşağıdaki adımlardan. 75 ° C'de 2 min için koşul contalar 60 ° C'de sonda tanıtmak Sonda ve örnekleri 5 dk 90 ° C'de denatüre 60 ° C'de 3 h için örnekleri melezlemek<ol>
<li>Slaytları 2 X SSC, % 0,1 (w/v) SDS, akış 10 s ile iki kez 50 ° C'de yıkama ve 20 s. 0.1 X SSC, % 0,1 SDS, akış 10 s ile iki kez 42 ° C'de yıkama slaytlar ve iki kez 0.1 ile 42 ° C'de 20 s. yıkama slaytlar tutun tutun X SSC , akış 10 s ve tutun 20 s.</li>
		</ol>
</li>
	</ol>
</li>
	<li>Kuru slayt Santrifüjü (olduğu gibi 4.6) tarafından.</li>
</ol>6. dizi miktar
<ol><li>İnce bir plastik film kullanarak slaytları sarın. Bir gayger sayacı onun en hassas ayarı kullanarak radyoaktif sinyali slaytlarını denetleyin. Slaytlar bir pozlama kaset, oda sıcaklığında 10-90 h bağlı olarak sinyal gücü için bir depolama fosfor ekrana maruz. Not: Geiger sayaçları duyarlılığını farklı olarak bir enstrüman diğerine, pozlama süreleri ampirik olarak optimize edilmiş olması gerekir.</li>
	<li>Slayt bir phosphorimager kullanarak 50 µm çözünürlükte tarama. Ölçmek ve radyoaktivite Koyulukları Mikroarray profiler ile yükseltilmiş ücretsiz görüntü J yazılımını kullanarak her sonda spot arka plan çıkarma.</li>
	<li>Düzenleyebilir ve bir elektronik tablo kullanarak verileri işlemek. Koşullu biçimlendirme aracını kullanarak ısı haritaları oluşturmak. Örneğin her sonda sinyal (‰ ifade edilir) bir kesir olarak toplam sonda sinyalinin temsil eder.</li>
</ol>

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Hiçbir çıkar çatışması ilan etti.

Ürünü test aralığındaki hücre fitness üzerinde önemli etkisi yoktur ancak beta parçacık emisyonları bakteriyostatik ve bakteri yok edici ajanlar14, bilinir. Mercek sayma radyoaktivite toplam-hücre özleri % 25'i ve daha sonra saf toplam RNA'ların % 1'i dahil gösterdi.
Probları aynı boyutlarda, karşılaştırılabilir erime sıcaklığı ve GC içeriğiyle macroarray lekelenme için Tekdüzen bir protokol hibridizasyon ve miktar tarafsız tRNA düzeylerinden spot sinyalleri doğrudan ölçümü için sağlar.
Tekrarlanabilir ve belirli bir noktadır. Onun geniş dinamik alan ve kolayca ayarlanabilir eşiği düşük gibi bol türlerin, sadece dizi pozlama kez9uzatan tarafından polizomlar ile ilişkili tRNA profil oluşturma sağlar. Sonra ilk yatırım için gerekli ekipman, sarf malzemeleri, dahil olmak üzere örnek maliyet çalışan $15-de örnekleri ortalamasını alır.
Nokta olan genom araştırma tasarımı için kullanılabilir herhangi bir organizma için geçerlidir. Vitro yetiştirilmektedir model organizmalar metabolik etiketleme için ideal aday olur. Memeli genleri genellikle birçok isodecoders (tRNA aynı anticodons paylaşmak, ancak onların vücut sekanslarında küçük farklılıklar görüntülemek) kodlamak gibi probları yakın tRNA türler homojen hibridizasyon korumak için kısmen dejenere olması gerekir. Son olarak, yapisan memeli hücreleri toplam RNA kültürleri önemli ölçüde ölçeklendirilmesi gereken süspansiyon gibi M. smegmatis, sonrasını organizmalar göre genellikle daha düşük miktarlarda verim.

Hücre tipi, çevre ve stres1,2,3gibi koşullara göre hücresel tRNA seviyeleri dalgalanma. Nokta, tRNA, gözlemlemek için aerodinamik Platform bir özgün, güvenilir ve laboratuvar yetiştirilen organizmalar transfer RNA düzeylerinin hızlı ve hassas miktar sağlar basit tekniği dir.
tRNA son derece kararlı ikincil ve üçüncül yapıları4var. Bunlar da çok sayıda çoğu değişiklikler5görüntüler. Bu özellikler önemli yapısal ve kutuplama ya da bazen doğrudan ve tekrarlanabilir standart kıyaslama RNA miktar teknikleri6kullanarak tRNA profil oluşturma önleyerek sıra yollara barikat temsil eder. Araştırma grupları dünya çapında hücresel tRNA düzeylerini değerlendirmek için yaratıcı ama kez kıvrık teknikleri geliştirmek zorunda kaldılar. Bu yöntemlerden bazıları şunlardır: (1) iki boyutlu elektroforetik ayrımı metabolik olarak etiketli tRNA kombine metodik nokta atama ile Kuzey leke1tarafından; (2) Kuzey tarafından bireysel miktar leke dikkatle standart radyoaktif DNA probları7kullanarak; (3) sonrası ayıklama siyanür fluorochromes ile etiketleme ardından Mikroarray analiz3ve (4) vitro rekombinant demodification enzimler ters transkripsiyon ile kombine ve sonraki kullanarak tRNA değişiklikler sıyırma yüksek işlem hacmi sıralama8.
Basit ve özgün birleşimi iki standart ve basit yaklaşım bir noktadır: organizmalar, radyoaktif toplam RNA'ların ve (2) tRNA macroarrays, sistematik ve kırılan genomik aracı büyüyen sentez, oluşan etiketleme, (1) RNA vücut tRNA ayrımı için en iyi duruma getirilmiş.
Örnekler canlı organizmalar miktar platforma aerodinamik. Onlar doğrudan çıkarma sonra analiz edilir ve önyargıları sonrası ayıklama amplifikasyon veya enzimatik tedavi gerektiren teknikleri ile karşılaştırıldığında önemli ölçüde sınırlayan daha fazla işlenen değil. Nokta çok yönlü ve kolayca tanımlamanıza ve ribozomlar çeviri ile fiziksel olarak ilişkili tRNA altgrupları ölçmek için polysome ayırma için kombine edilebilir.
Burada sunulan protokolü Mycobacterium smegmatisiçin optimize edilmiş ve o da başarıyla profil tRNA Escherichia coli9, Saccharomyces cerevisiae10, fare ve insan hücre kültürleri11 için kullanılan .

<table><tbody><tr><td>Glass slide indexing system</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 470</td><td></td></tr><tr><td>Glass slide arrayer replicator</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 478</td><td></td></tr><tr><td>96 well microplate indexing system</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 472</td><td></td></tr><tr><td>Wash and blot station</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 475</td><td></td></tr><tr><td>Replicator pin dryer</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP 474</td><td></td></tr><tr><td>Amine coated slides</td><td>Molecular Devices</td><td>K2615</td><td></td></tr><tr><td>Hybridizer / Automated hybridization and washing station</td><td>Digilab</td><td>Hyb10022</td><td></td></tr><tr><td>Shaker incubator</td><td>Eppendorf Themomixer</td><td>05-400-200</td><td></td></tr><tr><td>Screw cap 2 ml tubes</td><td>Thermo Scientific</td><td>21-403-201</td><td></td></tr><tr><td>[32P] Na2HPO4</td><td>Perkin Elmer</td><td>NEX011001MC</td><td></td></tr><tr><td>Guanidinium thiocyanate-phenol-chloroform (Trizol)</td><td>Invitrogen</td><td>15596026</td><td></td></tr><tr><td>0.5 mm glass beads</td><td>Research Products International Corp.</td><td>9831</td><td></td></tr><tr><td>Mini bead mill</td><td>VWR</td><td>25-020</td><td></td></tr><tr><td>Storage phosphore screen</td><td>Fujifilm</td><td>BAS-IP SR 0813</td><td></td></tr><tr><td>Phosphorimager</td><td>GE Healthcare</td><td>Typhoon FLA7000</td><td></td></tr><tr><td>DNA oligonucleotides</td><td>IDT</td><td>N/A</td><td></td></tr><tr><td>UV crosslinker</td><td>Spectroline</td><td>Select series 254 nm</td><td></td></tr><tr><td>Microarray centrifuge</td><td>Arrayit Corporation</td><td>MHC110V</td><td></td></tr><tr><td>&lt;em&gt;Mycobacterium smegmatis&lt;/em&gt;</td><td>ATCC</td><td>700084</td><td></td></tr><tr><td>Single-use needles</td><td>BD (Becton Dickinson)</td><td>305185</td><td></td></tr><tr><td>2 ml centrifuge tube</td><td>Fisherbrand</td><td>14-666-317</td><td></td></tr><tr><td>Precipitant (GlucoBlue)</td><td>Invitrogen</td><td>AM9516</td><td></td></tr><tr><td>7H9 broth</td><td>Difco</td><td>271310</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Chemicals</td><td>C298-500</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Chemicals</td><td>A451-4</td><td></td></tr><tr><td>20 X SSC</td><td>Lonza</td><td>51205</td><td></td></tr><tr><td>SDS</td><td>Fisher Bioreagents</td><td>BP166-100</td><td></td></tr><tr><td>hybridisation cassette</td><td>GE Healthcare</td><td>63-0035-44</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Tween 80</td><td>Amresco</td><td>M126-100ML</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Albumin</td><td>Spectrum Chemicals</td><td>A3611</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Chemicals</td><td>D16-1</td><td></td></tr><tr><td>EDTA (Ethylenediaminetetraacetic Acid)</td><td>Fisher Bioreagents</td><td>BP2482-500</td><td></td></tr><tr><td>0.5 M Phosphate buffer pH 7.2</td><td>Alfa Aesar</td><td>AAJ63974AP</td><td></td></tr></tbody></table>

metabolic labeling and profiling of transfer rnas using macroarrays

Transfer RNA (tRNA) genellikle 76-90 nükleotid uzunluğunda olan bol kısa kodlamayan RNA türü bulunmaktadır. tRNA, mRNA içinde kodon amino asit dizileri haline çevirerek protein sentezi için doğrudan sorumludur. tRNA uzun düzenleyici fonksiyonları yoksun ev tutma molekül olarak kabul edildi. Ancak, kanıt büyüyen bir vücudun hücresel tRNA düzeyleri değişen koşullara hücre tipi, çevre ve stres gibi yazışma dalgalanma gösterir. TRNA ifade dalgalanma lehine veya ifade belirli proteinlerin bastırmak gen çeviri, doğrudan etkiler. Sonuçta protein sentezi dinamik kavrama yöntem yüksek kaliteli tRNA profilleri teslim etmek mümkün geliştirme gerektirir. Burada mevcut yöntemi için aerodinamik Platform için gözlem tRNA standları yer almıştır. Hücre kültürleri radyoaktif orthophosphate, radyoaktif toplam RNA'ların guanidinium thiocyanate-fenol-kloroform çıkarma tarafından takip ile metabolik etiketleme ile başlayan üç adım nokta oluşur ve nihayet hibridizasyon in-house Tarih baskılı macroarrays. tRNA düzeyleri her sonda spot radyoaktivite Koyulukları miktarının tarafından tahmin edilir. Burada sunulan protokolünde Mycobacterium smegmatis mc2' de 155, sık sık bir model organizma tüberküloz çalışırdım nonpathogenic bir bakteri tRNA profil.

Üç bağımsız biyolojik, test büyüme koşullarda tüm M. smegmatis tRNA arka plan seviyesinden (şekil 1) ifade edilir göstermek çoğaltır. Belirli bu deneyde %2 (Arg (TCT)) ve medyan (şekil 1) % 5 ile % 22 oranında (Cys (GCA)) arasında gözlenen standart sapma her sonda için oluşur. Yanlış değişiklikleri çoğaltır arasında önemli ölçüde dizi hazırlık ve hibridizasyon gibi faktörlerden etkilenmektedir. Gibi örnek hibridizasyon otomatik yüksek tekrarlanabilirlik dikkatli ve tutarlı dizi baskı yoluyla elde edilir. TRNA arasında 5-fold fark Ile (GAT) ve tRNA (Val (TAC), en yüksek ve en düşük bol türler anılan sıraya göre. Nokta tRNA isoacceptors ifade tek tip olmadığını gösteriyor. Isoacceptors aynı amino asit tutun ancak farklı antikodon sıralarını görüntülemek ve bu nedenle farklı kodon mRNA'ların üzerinde kod çözme türü bulunmaktadır. Örneğin, tüm alanin isoacceptors benzer düzeyde ise ifade edilir kabul tRNA farklı tarafından 3-fold en yüksek ve en düşük arginin.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56898/56898fig1.jpg"> Şekil 1: temsilcisi tRNA macroarray sonuçları. (A) taranan bakteriyel, fare ve insan macroarrays. M. smegmatis diziler sadece 43 Probe prob 48 yerine fare ve insan için kullanın. Sonuç olarak, bakteriyel dizi arka plan ile Harman (probları her dizi üzerinde sekiz kez yinelenir) 40 boş noktalar görüntüler. Bunların çoğu sol üst köşesinde kümelenir. (B) çubuk grafik ve heatmap temsilleri M. smegmatis tRNA profillerinde optimal büyüme koşullar altında. Sonda sinyalleri üç bağımsız deneyler (dahil Bakteriyel büyüme, metabolik etiketleme, ayıklama ve dizi hibridizasyon) ortalama ve toplam tRNA bin değerleri başı olarak ifade edilir. Üç çoğaltır için standart sapmalar hata çubukları ve çubuk grafik ve heatmap turuncu tonları olarak sırasıyla belirtilmiştir. <a href="http://cloudflare.jove.com/files/ftp_upload/56898/56898fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>

Watch this Scientific Journal Video about Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays at JoVE.com

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Biz orijinal bir transfer RNA analizi platformu nokta (akıcı tRNA gözlemlemek için Platform) adlı tanımlamak. Nokta aynı anda hücresel düzeyde tüm tRNA biyolojik örnekleri, sadece üç adım ve 24 saatten az bir süre ölçer.

Metabolik etiketleme ve Transfer RNA Macroarrays kullanarak profil oluşturma

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

metabolic-labeling-and-profiling-of-transfer-rnas-using-macroarrays

Research

JoVE Journal

Genetics

5.8K Görüntüleme.  College of Charleston. Biz orijinal bir transfer RNA analizi platformu nokta (akıcı tRNA gözlemlemek için Platform) adlı tanımlamak. Nokta aynı anda hücresel düzeyde tüm tRNA biyolojik örnekleri, sadece üç adım ve 24 saatten az bir süre ölçer.

Video: Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

Yüksek Verimli MicroRNA Profil: Optimize edilmiş, Multiplex QRT-PCR Fluidigm Dinamik ArrayTM IFCS Nanoliter Ölçeğinde

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Biyomühendislik

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Gerçek zamanlı analiz bağlama, transkripsiyon, çeviri ve ciro hücresel etkinleştirme sırasında küresel olayları görüntülemek üzere transkripsiyon faktörü

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Genetik

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabolik 4-thiouracil ve bir miktar ile etiketleme yeni mRNA RNA polimeraz II etkinlik için bir Proxy olarak sentezlenmiş

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

RNA In Vivo'nun 4-Thiouracil (Ers4tU) ile Son Derece Hızlı ve Spesifik Metabolik Etiketlemesi

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Biyokimya

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Meme Kanseri Hücrelerinde östrojen düzenlenmiş microRNA profil

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Tıp

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

İnsan pre-mRNA üzerinde Haritalama Ribonucleoproteins Hızlı Yüksek verim için Yöntemi (RNPs)

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Biyoloji

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Kantitatif Gerçek-zamanlı PCR (qPCR) Diziler kullanarak Pre-mikro RNA&#39;lar ve microRNA, Profilleme

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

Hücre Kültürü RNA sentezi, İşleme ve Çürüme Yüksek Çözünürlük Gen İfadesi Profil için Yeni Transkripsiyonu RNA Metabolik Etiketleme

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polizom Profil Yöntemi ile memeli hücrelerine selektif mRNA dönüşümüyle Değerlendirilmesi

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR.
This video introduces the concepts of metabolic labeling and show two general procedures.&#160; The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos.

<div class="chapter_text" id="script_0">
Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications.
</div>
<div class="chapter_text" id="script_1">
Metabolic labeling can be conducted using a number of strategies. Here we will describe isotopic, photoreactive, and bio-orthogonal labeling.
Isotopic labeling is performed using structural analogs that are chemically identical to their natural counterparts, but have uncommon isotopes incorporated into their structure. In this L-lysine analog the carbon and nitrogen atoms are replaced with carbon-13 and nitrogen-15. Cells cultured in the presence of isotopic analogs will incorporate them into their biochemical structures. Metabolites are collected from the cells and purified for analysis. Samples with stable isotopes are analyzed using techniques such as mass spectrometry or NMR spectroscopy. Samples with radioactive labels are analyzed using liquid scintillation counting and x-ray films, which will be demonstrated in the isotopic labeling protocol.
Photoreactive labels are functional groups incorporated into proteins, which are stable until exposed to ultraviolet light. The functional group forms a reactive radical, which binds to the nearest protein. A common example, L-photo-leucine, contains a diazirine ring, which is a photoreactive crosslinker. In contrast to isotopic labeling, there is some chemical dissimilarity between photo-reactive chemical analogs and their natural counterparts. Cells may preferentially incorporate natural compounds over analogs. Therefore, it is important to perform photo-reactive labeling in medium free of the compound being mimicked. Once exposed to ultraviolet radiation, the photo-reactive groups in a labeled protein become unstable and highly reactive, causing it to cross-link with interacting proteins, creating a protein complex. Cross-linked complexes, act as snapshots that can then be analyzed using SDS-PAGE and mass spectrometry methods. This provides insights into what reactions are occurring in the metabolic pathway by identifying reaction species and how they interact by determining binding sites.
Bioorthogonal labeling strategies utilize analogs with small functional groups that have little to no reactivity with natural biomolecules. For example, azides are small functional groups, whose reactivity is said to be orthogonal to biochemical reactions. In the Staudinger ligation, a phosphine group attacks the azido group. This yields a transition state that intramolecularly reacts with a nearby ester, resulting in an amine-bonded ligand. Bioorthogonal functional groups incorporated into biomolecules can be ligated with detection tags such as fluorescent functional groups, and affinity tags such as antigens.
Now that some concepts and strategies for metabolic labeling have been discussed, let&#39;s look at the process in the laboratory.
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The first step in a metabolic labeling experiment is to collect the protein of interest. To do this, cells are grown on a plate, and an expression method is used to promote synthesis of the desired protein. In this example, leucine-rich repeat kinases, or LRRK, are expressed. Disodium phosphate, containing radioactive phosphorus-32, is used as the analog. Proper measures must be taken to protect against ionizing radiation. This includes setting up a work area, wearing proper protective equipment, and checking for radioactive contamination. Once safety measures have been taken, the medium containing the isotopic analogs is prepared. The medium from the culture is removed and, replaced with one containing the isotopic chemical analogs and then incubated. Following incubation, the cells are lysed. The lysate is collected and purified.
After purification, proteins are resolved using SDS-PAGE and then transferred to a PVDF membrane. Autoradiography is performed by exposing the membrane to x-ray film and measured using a phosphor imager. Western blotting is used to measure relative protein levels in the PVDF membrane. In this example the phosphorylation levels of leucine-rich repeat kinases synthesized in 293T cells were measured. The autoradiogram shows how much phosphorous was incorporated into the protein. Western blotting elucidates the levels of the LRRK proteins. Image analysis software is used to obtain quantitative data of phosphorylation levels of the proteins.
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In this next procedure, photoreactive labeling is demonstrated. First, the cells are prepared and cultured. The photoreactive analog is added to the cells at the mid-log phase and incubated. In this procedure p-benzoylphenylalanine is used. Samples are collected over intervals and put on ice. The samples are then exposed to obtain snapshots of the biochemical pathways over time. The proteins of interest are then purified and resolved using SDS-PAGE.
A photoreactive labeling strategy was used to identify compounds that interact with the protein of interest. Immunodetection with Western blotting shows protein bands that indicate higher molecular weight proteins are present in the irradiated samples. These are from cross-linking due to protein-protein interaction occurring during the UV irradiation.
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Now that we&#39;ve reviewed metabolic labeling procedures, let&#39;s look at some of the ways the process is used.
Metabolic labeling concepts can be extended to multicellular organisms. Plants are grown in a sealed environment, rich in stable isotopes to produced labeled plant material. Carbon dioxide containing carbon-13 is added to the enclosure, while nitrogen-15 rich fertilizer is used. The resulting harvested plant material can help answer questions about carbon and nitrogen cycling from the ecosystem.
Labeling enables the separation of newly synthesized RNA from older RNA. By changing the initial concentration of analog, the kinetics of new RNA synthesis can be determined. The results show that the concentration of 4-thiouridine affects how much new RNA is transcribed. Additionally, incorporation rates of the label into RNA can be directly quantified with a spectrophotometer.
Using biorthogonal click chemistry, glycans in a zebra fish embryo can be labeled. The eggs are injected with a labeling compound that results in alkyne labels on the glycans. The glycans in the larvae are then ligated to a dye compound at the desired development stage. The glycans in the embryos are then imaged. Glycans produced at different time points can be identified by labeling using different colors at different stages of embryo development.
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You&#39;ve just watched JoVE&#39;s video on metabolic labeling. This video described the concepts behind metabolic labeling and their strategies, went over two general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Isotopic, Photoreactive and Bioorthogonal Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs ...