Biz L. Evans, B. Granger, D. Rissling, Z. Sim, A. Goodwin, K. Hayhurst ve D. Kuch alanında yardım için teşekkür etmek istiyorum. K. Galbreath lütfen Bella Coola vadiden pika karaciğer örnekleri sağladı. Bu noninvaziv genetik örnekleme yöntemi tasarımına katkıda ilginç tartışmalar için, A. Gonçalves Da Silva ve K. Larsen teşekkür ederiz. Bu çalışma, Doğal Bilimler ve Mühendislik Araştırma Konseyi Kanada Keşif ve MAR UBC Okanagan Bireysel Araştırma hibe tarafından finanse edildi. İsviçre Ulusal Bilim Vakfı Doktora Bursu PBSKP3_128523 PH desteklenir. Bu çalışma, British Columbia Üniversitesi (A07-0126 Sertifika numarası) hayvan bakım protokolü takip yapıldı.

1. Saç tuzak Bir tuzak kurmak için önce, ideal bir konuma pika habitat veya talus eğimi içinde belirlenecek. Bu hayvanlar yaz sonu gibi tuvaletleri sitelerinde bulunan taze scats topladığımız bitki örtüsü önbelleklerini saman yığınları içerir. Şeritler ambalaj bant (uzunluğu 10-50cm), 360 derecelik bir yapışkan bir yüzey sağlamak için alınır ve zarf pika saman yığını (Şekil 1) veya tuvalet sitenin girişinde bir web gibi bir şekilde düzenlenmiştir. Misina kayaların yapılandırmasına bağlı olarak, bir parça saç tuzak yapısını desteklemek için kullanılır, fakat bu girişleri, tam olarak bir açıklama kullanımı oldukça küçük (çapı &lt;30cm) çoğu zaman gerekli değildir. Henry ve Russello (2010) 3 misina bulunabilir. Saç tuzaklarına mümkün olduğunca sık kontrol ve bant (Şekil 2) saç örnekleri üzerinde biriken sonra toplanan ve etiketlenir. Laboratuara bir kez geri taşınır, saçlar daha fazla manipülasyon kadar steril forseps kullanarak yapışkan bandı kaldırılmış ve kriyojenik tüpler aktarılır ve -20 ° C&#39;de saklanır. Bağımsız kümelenmiş örnekleri farklı bireylere ait olduğu kabul edilir bir saç tuzak boyunca bir araya kümelenmiş Saç örnekleri, tek bir bireye ait olduğu kabul edilir. İkinci durumda, saç farklı tüpler yerleştirilir. Bu varsayımlar, daha sonra mikrosatellit genotipik verilere göre DNA parmak izi ve kimlik olasılık hesabı yoluyla test edilebilir. 2. DNA ekstraksiyonu Pika saç çok ince ve çok küçük bir kök ampul içerdiğinden, daha önce en az 25 kılların kaliteli downstream PCR 3 için yeterli miktarda DNA elde etmek için gerekli olduğunu göstermiştir . Biz DNA IQ sistemi (Promega, Madison, WI, USA) ve kıl örneklerinden DNA izolasyonu için üreticinin talimatlarına biraz değiştirilmiş bir versiyonu kullanıldı. Öncelikle, saç örneği tüp açarken saç kaybını önlemek amacıyla bir mikro santrifüj eğirilir. Daha sonra kuluçka çözüm Proteinaz K. Kuluçka 0.1 M DTT ve 1.8ng/ml bir final konsantrasyon neden DTT 10μl (1M) ve 10μl, proteinaz K (18ng/ml) ile 80 ul inkübasyon çözüm karıştırılarak hazırlanır solüsyonu (100 ul) örnek eklenir ve 56 ° C &#39;de 1 saat süreyle inkübe edilir. Bu arada, lizis çözüm 0.01m son bir DTT konsantrasyon, lizis tamponu her 100 ul DTT (1M) 1 ul ekleyerek hazırlanır. Hazırlanan lizis solüsyonu (200 ul), daha sonra 7 ul DNA IQ reçine ile birlikte, örnek, yüksek hızda 3 saniye için vortekslenmiş ve 5 dakika oda sıcaklığında inkübe. Örnek sonra 2 saniye boyunca vortekslenmiş ve manyetik boncuk çözüm ayrılması meydana gelen manyetik standı, takılmış. Sonra kalan çözüm emişli ve atılır, manyetik reçine pelet rahatsız etmemek için dikkatli olmak. Örnek sonra hazırlanan lizis çözüm vortekslenmiş ve ayrılık yeniden oluşursa manyetik stand döndü 100 ul ile tedavi edilir. Aspire lizis tamponu ve atılır ve bu adımı yıkama tamponu (100 ul) kullanarak üç kez tekrarlanır. Örnek daha sonra, 15 dakika boyunca manyetik stand kurumaya bırakılır. Kuruduktan sonra, 100 ul elüsyon tampon örnek eklenir ve 65 ° C&#39;de 5 dakika inkübe. Örnek vortekslenmiş ve manyetik stand içine eklenir. Yıkandı, DNA içeren bir çözüm daha sonra 1.5 ml Eppendorf tüpüne aktarılır ve daha fazla manipülasyonlar kadar -20 ° C&#39;de saklanır. Karaciğer örneklerinden DNA DNA IQ sistemi kullanılarak ayıklanır ve PCR için bir pozitif kontrol olarak kullanıldı. 3. PCR Noninvaziv saç tuzak ve karaciğer örnekleri elde edilen DNA, sonra yaygın olarak kullanılan moleküler belirteçlerin (mikrosatellit, AFLP, mitokondriyal sitokrom B ve ZFX / ZFY cinsiyet tayini işaretleyici) bir paketi yükseltmek için kullanılır. - 20 ng DNA, her astar 0.5 mcM, 10 mM Tris-HCl (pH 8.3), 50 mM 10: PCR içeren 25 mcL hacmi Veriti termal cycler (Applied Biosystems, Foster City, CA, USA) kullanılarak yapıldı KCl, 1.5 mM MgCl 2, 200 mcM dNTP, 10 μ sığır serum albumini (BSA; New England Biolabs, Ipswich, MA, ABD) ve 0.5 U AmpliTaq Gold DNA polimeraz (Applied Biosystems). Mikrosatellit lokus&#39;u Bisiklete binme parametreleri (95 10 dakika bir gol bisiklet programı kullanılarak optimize edilmiştir ° C, 95 ° C - 30 s, 30 s tavlama az 35 kür, 45 sn ve 72 ° C, 72 son bir adım ° C 10 dk). 1 azalarak tavlama ° C sıcaklık döngüsü başına 60 ila 55 ° C kalan 29 döngüleri 55 devam altıncı döngüsü, hangi noktada ° C ulaşana kadar Mitokondriyal parçaları için Bisiklete binme parametreleri yukarıda açıklandığı gibi ama bir tavlama ile 35 döngü oluşturdu50 ° C sıcaklık Amplifiye Fragment Uzunluk Polimorfizm Bonin 4 tarafından açıklanan omurgalı genomların için protokol yapıldı. Son olarak, moleküler cinsiyet tayini HinfI restriksiyon enzim sindirimi 5 ZFX / ZFY lokusların PCR-RFLP yöntemi kullanılarak yapıldı. Sindirilir ZFX / ZFX parçaları içeren% 3 agaroz jel üzerinde 100 baz puan (bp) merdiven (New England Biolabs) yanında çalıştırmak iken mikrosatellit AFLPs ve sitokrom B dizileri PCR ürünleri, ABI 3130XL genetik analizör (Uygulamalı Biyosistem) üzerine çalıştırmak % 2.5 SYBR Güvenli DNA jel leke (Invitrogen, Carlsbad, CA, USA) ve KIRMIZI kişisel bir jel görüntüleme sistemi (Alpha Innotech, San Leandro, CA, USA) kullanılarak görüntülenebilmekte. Mikrosatellit ve AFLPs Genemapper v3.7 (Uygulamalı Biyosistem) ve mitokondriyal DNA kromatogram kullanarak Sequencher v4.7 (Gen Kodu Corporation, Ann Liman, MI, USA) kullanılarak görüntülendi görüntülendi. 4. Temsilcisi sonuçları Bizim noninvaziv tuzak kullanılarak elde edilen saç örneklerinden çıkarılan DNA PCR moleküler belirteçler çeşitli tip yükseltmek için kullanılmıştır. DNA karşılaştırması için bir temel olarak karaciğer örnekleri saç örnekleri yanında amplifiye edildi kullanarak ayıklanır. Nükleer mikrosatellit lokus&#39;u saç ve karaciğer örnekleri (Şekil 3) hem de başarılı bir şekilde amplifiye edildi. Karaciğer örnekler kullanılarak sinyal yoğunluğu daha fazla olmasına rağmen, bu genotip puanlama üzerinde olumsuz bir etkisi yoktu (Şekil 3). AFLPs (Şekil 4), mitokondriyal DNA dizilerinin (Şekil 5) ve ZFX / ZFY moleküler cinsiyet tayini işaretleyici (Şekil 6) kullanarak benzer sonuçlar elde edildi. <img src="/files/ftp_upload/2791/2791fig1.jpg" alt="Şekil 1"/> Şekil 1 noninvaziv bir saç tuzak bir örnek, bir saman yığını. Kadar rulo ambalaj bant şeritler, saman yığını girişinde içine bir web gibi moda düzenlenmiştir. Misina bir parça saç tuzak için ek destek sağlamak için kullanılır. <img src="/files/ftp_upload/2791/2791fig2.jpg" alt="Şekil 2"/> Şekil 2&#39;de ambalaj bandı sıkışmış saç çok sayıda içeren bir başarıyla saç snare <img src="/files/ftp_upload/2791/2791fig3.jpg" alt="Şekil 3"/> Şekil 3 bir temsilci nükleer mikrosatellit kromatogram (Ocp6) 8 Genemapper v3.7 (Applied Biosystems) görüntülenir. Üst kromatogram karaciğer dokusu yükseltme DNA ile elde edilen ve bu lokus (354/358) heterozigot idi. Alt kromatogram saç çıkarılan DNA dayalı bir heterozigot genotip (358/362) sergileyerek farklı bir kişi temsil eder. Saç örneği kromatogram sinyal karaciğer örneği daha düşük bir yoğunluğu olsa da, genotip puanlama sinyal bu fark olumsuz etkilenmez. <img src="/files/ftp_upload/2791/2791fig4.jpg" alt="Şekil 4"/> Şekil 4 temsilcisi AFLP kromatogram (E31T32) Genemapper v3.7 (Uygulamalı Biosystems) görüntülenir. Üst kromatogram karaciğer dokusu yükseltme DNA elde edildi, alt kromatogram saç çıkarılan DNA temsil eder. <img src="/files/ftp_upload/2791/2791fig5.jpg" alt="Şekil 5"/> Şekil 5 Sequencher v4.7 (Gen Kod Corporation) görüntülenen bir temsilci, mitokondriyal DNA dizisi kromatogram (sitokrom B). Üst kromatogram karaciğer dokusu yükseltme DNA elde edildi, alt kromatogram saç çıkarılan DNA temsil eder. <img src="/files/ftp_upload/2791/2791fig6.jpg" alt="Şekil 6"/> Şekil 6, karaciğer ve saç örnekleri için yükseltilen temsili moleküler cinsiyet tayini işaretleyici (ZFX / ZFY) . Bu yaklaşım, Y kromozomu X kromozomu yoksun olduğunu, bu bölgede bir HinfI kısıtlama sitenin sahip olduğu gözlemine dayanır. Erkeklerde ise 432 bp, 261 bp ve 171 bp parçaları üreten Böylece, kadınlarda 432 bp iki ortak göç parçaları üretir.

<ol>
	<li>Kendall, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demography+and+Genetic+Structure+of+a+Recovering+Grizzly+Bear+Population.">Demography and Genetic Structure of a Recovering Grizzly Bear Population.</a> J Wildlife Manage. 73, 3-3 (2009).</li><li>Rudnick, J. A., Katzner, T. E., Bragin, E. A., DeWoody, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-invasive+genetic+evaluation+of+population+size,+natal+philopatry,+and+roosting+behavior+of+non-breeding+eastern+imperial+eagles+(Aquila+heliaca)+in+central+Asia.">A non-invasive genetic evaluation of population size, natal philopatry, and roosting behavior of non-breeding eastern imperial eagles (Aquila heliaca) in central Asia.</a> Conserv. Genet. 9, 667-667 (2008).</li><li>Henry, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+population+structure+and+ex+situ+representation+of+the+endangered+Amur+tiger.">In situ population structure and ex situ representation of the endangered Amur tiger.</a> Mol Ecol. 18, 3173-3173 (2009).</li><li>Henry, P., Russello, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+high+quality+DNA+from+elusive+small+mammals+using+low-tech+hair+snares.">Obtaining high quality DNA from elusive small mammals using low-tech hair snares.</a> Eur J Wildlife Res. , (2010).</li><li>Bonin, A., Pompanon, F., Taberlet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+amplified+fragment+length+polymorphism+(AFLP)+markers+in+surveys+of+vertebrate+diversity.+Method+Enzymol+395.">Use of amplified fragment length polymorphism (AFLP) markers in surveys of vertebrate diversity. Method Enzymol 395.</a> , 145-145 (2005).</li><li>Fontanesi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexing+European+rabbits+(Oryctolagus+cuniculus),+European+brown+hares+(Lepus+europaeus)+and+mountain+hares+(Lepus+timidus)+with+ZFX+and+ZFY+loci.">Sexing European rabbits (Oryctolagus cuniculus), European brown hares (Lepus europaeus) and mountain hares (Lepus timidus) with ZFX and ZFY loci.</a> Mol Ecol Resour. 8, 1294-1294 (2008).</li><li>Piggott, M. P., Taylor, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+collection+of+animal+DNA+and+its+applications+in+conservation+management+and+understanding+the+population+biology+of+rare+and+cryptic+species.">Remote collection of animal DNA and its applications in conservation management and understanding the population biology of rare and cryptic species.</a> Wildl. Res. 30, 1-1 (2003).</li><li>Banks, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demographic+monitoring+of+an+entire+species+(the+northern+hairy-nosed+wombat,+Lasiorhinus+krefftii)+by+genetic+analysis+of+non-invasively+collected+material.">Demographic monitoring of an entire species (the northern hairy-nosed wombat, Lasiorhinus krefftii) by genetic analysis of non-invasively collected material.</a> Anim. Conserv. 6, 101-101 (2003).</li><li>Litvaitis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+range-wide+survey+to+determine+the+current+distribution+of+New+England+cottontails.">A range-wide survey to determine the current distribution of New England cottontails.</a> Wildlife Soc B. 34, 1190-1190 (2006).</li><li>Peacock, M. M., Kirchoff, V. S., Merideth, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+nine+polymorphic+microsatellite+loci+in+the+North+American+pika,+Ochotona+princeps.">Identification and characterization of nine polymorphic microsatellite loci in the North American pika, Ochotona princeps.</a> Mol Ecol Notes. 2, 360-360 (2002).</li></ol>

Noninvaziv genetik örnekleme çeşitli nedenlerden dolayı geleneksel canlı yakalama yöntemleri için cazip bir alternatif haline gelmiştir. Birincisi, öne çıkmayan vahşi popülasyonlar biyolojik madde (örneğin, dışkı, tüyler, tüy, tükürük ve mukus) toplayarak, araştırmacılar, böylece hem hayvan ve araştırmacılar risklerin azaltılması, taşıma, hatta onları gözlemleyerek, rahatsız etmeden bu tür eğitim görebilirsiniz. İkincisi, zor ve nadir türlerin popülasyonlarında, daha geleneksel canlı tutucu yaklaşımları 6 ile zor bir görev noninvaziv genetik örnekleme çalışma biyologların sağlar. Ve üçüncüsü, NGS potansiyel böylece popülasyon parametreleri 7 tahminleri önyargıları en aza indirmek için yardımcı rahatsızlık hayvanlar, numune alma çabaları ve maliyetleri azaltarak, örnek boyutlarını artırabilir. Tehdit altındaki türler ile uğraşırken nüfus parametreleri önyargılı tahminler uygunsuz yönetimi neden olabilir Bu son nokta, önemli olabilir. Mevcut video makalede biz, bir örnek olarak Amerikan Pika, zor küçük memeliler örnek, basit, roman, ucuz ve noninvaziv bir yöntem kullanarak, açıklanmaktadır. Biz, bu non-invaziv örnekleme yöntemi, yakalama ya da ölümcül örnekleme yaşamak için cazip bir alternatif haline saç çıkarılan bu DNA mikrosatellit, AFLP, mitokondriyal ve cinsiyet tayini belirteçler açısından karaciğer örneklerden çıkarılan DNA benzer şekilde gerçekleştirir göstermektedir. Genel olarak, çalışmanın altında canlılar üzerindeki etkisini en aza indirirken, bu yöntem, nadir veya zor küçük memeli türlerin genetik ve davranışsal çalışmalar nüfus veri toplama aşamasında yararlı olacağını tahmin ediyoruz.

Saç tuzak: <ul><li> Net, ambalaj bandını Rolls (3M, St Paul, MN, ABD) </li><li> Misina (£ 10) </li><li> Kriyojenik tüp (2ml) </li><li> Steril forseps </li><li> Sıvı Azot Dewar (isteğe bağlı) </li></ul> DNA ekstraksiyonu: <ul><li> Doku ve saç çıkarma Kiti (Promega Kat. # DC6740), DNA IQ Sistemi (Promega Kat. # DC6701). </li><li> MagneSphere Teknoloji Manyetik Ayırma Standı (Kat. # Z5342; Promega) </li><li> Mikro santrifüj </li><li> Hibridizasyon fırın veya su banyosu </li><li> 1.5 ml Eppendorf Tüpleri </li></ul>

a noninvasive hair sampling technique to obtain high quality dna from elusive small mammals

Noninvaziv genetik örnekleme yaklaşımları yaban hayatı popülasyonları çalışma için giderek daha önemli hale gelmektedir. Bir dizi çalışmada, popülasyon genetiği ve vahşi popülasyonlar 1 demografi araştırmak için non-invaziv örnekleme teknikleri kullanılarak bildirdin . Bu yaklaşım, nadir veya zor türlerin 2 ile uğraşırken özellikle yararlı olduğu kanıtlanmıştır . Bu yöntemlerden bir dizi örnek saç, dışkı ve diğer etobur ve orta ölçekli memelilerin biyolojik madde için geliştirilmiş olsa da, büyük ölçüde zor küçük memeliler denenmemiş kaldı. Bu videoda, bir roman, ulaşılması zor bir küçük memeli, Amerikan pika (Ochotona princeps&#39;in) hedef ucuz ve noninvaziv saç tuzak mevcut. Biz web benzeri bir biçimde düzenlenmiş ve pikas &#39;yaşam alanı seyahat rotaları boyunca yerleştirilen bant ambalaj şeritleri oluşan saç tuzak, set-up genel açıklar. Biz büyük bir miktar sonra toplanır ve laboratuara geri getirdi saç toplama tuzak etkinliğini göstermektedir. Daha sonra DNA izole etmek ve nükleer Mikrosatellitlerin amplifiye parça uzunluk polimorfizmi (AFLPs), mitokondriyal dizileri (800bp) yanı sıra da dahil olmak üzere yaygın olarak kullanılan moleküler belirteçler yükseltmek için bu yöntemi, yardımcı vitrin DNA IQ sistemi (Promega) kullanımını göstermek bir moleküler cinsiyet tayini işaretleyici. Genel olarak, yaban hayatı nüfus biyologlar için bir örnekleme tekniği olarak bu romanı noninvaziv saç tuzak yararını göstermektedir. Biz bu yaklaşımın doğal popülasyonları içinde çalışma organizmaların etkilerini en aza indirerek, araştırma alanları açılması, küçük memeliler çeşitli için geçerli olacağını tahmin ediyoruz.

Watch this Scientific Journal Video about A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals at JoVE.com

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Biz Amerikan pika gösterildiği gibi, verimli bir şekilde zor küçük memeliler saç örnekleri toplamak için bir non-invaziv örnekleme yaklaşımı sunuyoruz. Biz örneklenmiş saç DNA açılan ve yaban hayatı ekolojisi ve korunması çalışmalarında yaygın olarak kullanılan moleküler belirteçler çeşitli yükseltme bu yöntemin yararını göstermektedir.

Elusive Küçük Memeliler Yüksek Kalite DNA Edinme Noninvaziv Saç Örnekleme Tekniği

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

a-noninvasive-hair-sampling-technique-to-obtain-high-quality-dna-from

Research

JoVE Journal

Biology

20.7K Görüntüleme.  University of British Columbia, Okanagan Campus. Biz Amerikan pika gösterildiği gibi, verimli bir şekilde zor küçük memeliler saç örnekleri toplamak için bir non-invaziv örnekleme yaklaşımı sunuyoruz. Biz örneklenmiş saç DNA açılan ve yaban hayatı ekolojisi ve korunması çalışmalarında yaygın olarak kullanılan moleküler belirteçler çeşitli yükseltme bu yöntemin yararını göstermektedir.

Video: A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

İnsan Postmortem Beyin Dokusu Tek Hücre Popülasyonlar Yüksek Kalite RNA Alınması

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Biyoloji

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Noninvaziv In Vivo Küçük Hayvan MRG ve MRS: Temel Deneysel Prosedürler

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Hitachi F-7000 Floresans Spektrofotometre ve PicoGreen Boya kullanarak dsDNA Niceleme

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA Mikroarray&#39;ler: Örnek Kalite Kontrol, Dizi Hibridizasyon ve Tarama

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Mikrobiyal Paspas Yüksek Moleküler Ağırlıklı DNA çıkarımı

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Toz numuneler mutlak Kuantum Verimi Ölçümü

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Yüksek verim Sıralama için Yüksek Kalite Kromatin Immunoprecipitation DNA Şablon üretimi (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebra balığı Embriyolar kullanılarak Yüksek verim Yaklaşıyor A Manuel Küçük Molekül Ekran

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Kanatlı Üretim Örneklerden Bakteriyel Topluluklar Nitel ve Nicel Değerlendirme Karma DNA Ekstraksiyon Yöntemi

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Rat Yanal Kuyruk Ven gelen Kan Örnekleme

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...