Bu eser diyabet Kanada tarafından desteklenmiştir.

<ol>
	<li>Guh, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incidence+of+co-morbidities+related+to+obesity+and+overweight:+a+systematic+review+and+meta-analysis.">The incidence of co-morbidities related to obesity and overweight: a systematic review and meta-analysis.</a> BMC. Public Health. 9, 88 (2009).</li><li>Wronska, A., Kmiec, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+biochemical+characteristics+of+various+white+adipose+tissue+depots.">Structural and biochemical characteristics of various white adipose tissue depots.</a> Acta Physiol (Oxf). 205 (2), 194-208 (2012).</li><li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue:+function+and+physiological+significance.">Brown adipose tissue: function and physiological significance.</a> Physiol Rev. 84 (1), 277-359 (2004).</li><li>Holland, N. T., Smith, M. T., Eskenazi, B., Bastaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+sample+collection+and+processing+for+molecular+epidemiological+studies.">Biological sample collection and processing for molecular epidemiological studies.</a> Mutat Res. 543 (3), 217-234 (2003).</li><li>Ibrahim, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+and+visceral+adipose+tissue:+structural+and+functional+differences.">Subcutaneous and visceral adipose tissue: structural and functional differences.</a> Obes Rev. 11 (1), 11-18 (2010).</li><li>Mann, A., Thompson, A., Robbins, N., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization,+identification,+and+excision+of+murine+adipose+depots.">Localization, identification, and excision of murine adipose depots.</a> J Vis Exp. (94), (2014).</li><li>Hemmrich, K., Denecke, B., Paul, N. E., Hoffmeister, D., Pallua, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Isolation+from+Adipose+Tissue:+An+Optimized+Procedure+for+High+RNA+Yield+and+Integrity.">RNA Isolation from Adipose Tissue: An Optimized Procedure for High RNA Yield and Integrity.</a> Laboratory Medicine. 41 (2), 104-106 (2010).</li><li>Cirera, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+method+for+isolation+of+total+RNA+from+adipose+tissue.">Highly efficient method for isolation of total RNA from adipose tissue.</a> BMC Res Notes. 6, 472 (2013).</li><li>Tan, S. C., Yiap, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA,+RNA,+and+protein+extraction:+the+past+and+the+present.">DNA, RNA, and protein extraction: the past and the present.</a> J Biomed Biotechnol. 2009, 574398 (2009).</li><li>Sellin Jeffries, M. K., Kiss, A. J., Smith, A. W., Oris, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+commercially-available+automated+and+manual+extraction+kits+for+the+isolation+of+total+RNA+from+small+tissue+samples.">A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples.</a> BMC Biotechnol. 14, 94 (2014).</li><li>Tan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+prorenin/renin+receptor+on+the+development+of+obesity+and+associated+cardiometabolic+risk+factors.">Impact of the prorenin/renin receptor on the development of obesity and associated cardiometabolic risk factors.</a> Obesity (Silver. Spring). 22 (10), 2201-2209 (2014).</li><li>Taylor, S., Wakem, M., Dijkman, G., Alsarraj, M., Nguyen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+RT-qPCR-Publishing+data+that+conform+to+the+MIQE+guidelines.">A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines.</a> Methods. 50 (4), 1-5 (2010).</li><li>Aranda, P. S., LaJoie, D. M., Jorcyk, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleach+gel:+a+simple+agarose+gel+for+analyzing+RNA+quality.">Bleach gel: a simple agarose gel for analyzing RNA quality.</a> Electrophoresis. 33 (2), 366-369 (2012).</li><li>Mercure, C., Prescott, G., Lacombe, M. J., Silversides, D. W., Reudelhuber, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+increases+in+circulating+prorenin+are+not+associated+with+renal+or+cardiac+pathologies.">Chronic increases in circulating prorenin are not associated with renal or cardiac pathologies.</a> Hypertension. 53 (6), 1062-1069 (2009).</li><li>Dusaulcy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-specific+disruption+of+autotaxin+enhances+nutritional+fattening+and+reduces+plasma+lysophosphatidic+acid.">Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.</a> J. Lipid Res. 52 (6), 1247-1255 (2011).</li><li>Nakamura, K., Fuster, J. J., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipokines:+a+link+between+obesity+and+cardiovascular+disease.">Adipokines: a link between obesity and cardiovascular disease.</a> J Cardiol. 63 (4), 250-259 (2014).</li><li>Taylor, S. C., Mrkusich, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+RT-quantitative+PCR:+firsthand+observations+of+implementation+of+minimum+information+for+the+publication+of+quantitative+real-time+PCR+experiments+(MIQE).">The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE).</a> J Mol Microbiol Biotechnol. 24 (1), 46-52 (2014).</li><li>Hummon, A. B., Lim, S. R., Difilippantonio, M. J., Ried, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+solubilization+of+proteins+after+TRIzol+extraction+of+RNA+and+DNA+from+patient+material+following+prolonged+storage.">Isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from patient material following prolonged storage.</a> Biotechniques. 42 (4), 467-470 (2007).</li></ol>

Yazarlar ifşa gerek yok.

Aşağıdaki HF-diyet, beslenme obez fareler vücut ve beyaz yağ dokusu ağırlık N diyet beslenen fareler için karşılaştırıldığında artmıştır bulunmuştur. Fenol çözüm kullanarak çıkarılan RNA iyi saflığı ile örnekleri vermiştir. Leptin öncelikle yağ dokusu tarafından üretilen bir adipokine ve olumlu fat kitle16ile ilişkilendirmek için bilinir. Beklendiği gibi leptin mRNA ifade obez farelerde concomitance onların yağ kitlesi ile artmıştır.
Bu yöntem birkaç önemli adımlar vardır. Farklı yağ dokusu depoları farklı işlevleri2,5var bilinen büyük kirlenme bir beyaz yağ dokusu depo başka bir ile olanıdır. Kirlenme downstream uygulamalarında yanıltıcı sonuçlar getirebilir. Şişman fareler arasında düzenli ve tutarlı bir hızda beyaz yağ dokusu toplama hava ile temas halinde bir kez kuru eğilimi gibi Ayrıca, kilo alınması gerekip gerekmediğini önerilir. Aksi takdirde, bir yağ yastığı toplam ağırlığı yanlış olabilir. Kısa bir süre önce MIQE takip RNA iyi kalite ve verim, almak için kurallar olduğunu kesin kıymet17. Özellikle, taşlama sırasında çok iyi örnek toz verme verimi en üst düzeye çıkarmak yardımcı olabilir. Bu RNA izolasyon sırasında doku toz ve fenol çözüm arasında temas en üst düzeye çıkarır. Son ama en az değil RNA içeren katman yalıtım sırasında RNA izolasyon (Adım 3.12) olduğunu. Yağ daha az su daha yoğun olduğu için faiz faz üzerine yerleştirilir. Yağ etkilenmişimdir en aza indirme aşağı akım uygulamaları ile girişim azaltmak gereklidir.
Bu yöntemin bir sınırlama yordamı hem de RNA izolasyon sırasında zararlı Kimyasalları ile çalışmak için gereken birkaç adımları gerçekleştirmek için gerekli beceri olduğunu. Ayrıca, tüm emek yoğun bir süreçtir.
Yağ dokusu toplama ve RNA izolasyon genellikle her kullanıcı tarafından ayarlanır küçük ayrıntılar dışında önce işleme örnek yöntemine çok alternatif değildir. RNA izolasyon söz konusu olduğunda, fenol/kloroform ayıklama veya RNA izolasyon kitleri gibi birçok seçenek mevcuttur. Orada avantajları ve dezavantajları her seçenek ve aşağı akım uygulamaları esaslı en iyi yöntemi seçmesini kalmıştır. Fenol/kloroform ayıklama daha ucuz ama zararlı Kimyasalları kullanımı gerektirir ve zahmetli. RNA izolasyon kitleri genellikle daha pahalıdır ama prosedür daha hızlıdır ve genellikle iyi bir kalite ve saflık örnekler verir. Malzeme beyaz yağ çoğunlukla oluşan dokusunda yağ sınırlı olması nedeniyle verim ve RNA saflığı dikkate almak önemlidir. Fenol çözüm için yalıtım (fenol/kloroform çıkarma) kullanarak önemli yararı RNA, DNA ve protein bir tek örnek18ayırma imkanı vardır. Bu maliyet ve zaman etkili gibidir yeterli malzeme almak için gerekli fare sayısını azaltır. Daha önce de belirtildiği gibi yağ kitlesi bazı fare modellerinde sınırlıdır. Bu aşağı akım uygulamalar için yetersiz malzeme neden gibi yere yağ dokusu ayrı ayrı RNA, DNA ve protein elde etmek için üç parçaya bölerek bu fareler için önerilmez. Bu sorunu aşmak için birlikte gerekli hayvan sayısının bir artmasına yol benzer yağ dokusu perondan kullanıcı tanımlı kriterlere göre birkaç fare havuzu mümkündür.

Obezite tip 2 diyabet1gibi komplikasyonlara yol açabilir bir dünya çapında salgın var. Obezite ve onun ilişkili komplikasyonlar diyet kaynaklı obez ve genetiği değiştirilmiş hayvan modelleri araştırma için sık kullanılır. Geleneksel olarak, beyaz yağ dokuları aşırı enerji için bir saklama bölmesi olarak bilinir ve kahverengi yağ dokusu enerji ısı2,3içine dönüştürünceye çoğunlukla lipidler oluşur. Yağ dokusu dinamiktir ve genişletilir ve gıda alımı ve fiziksel aktivite gibi birçok faktöre bağlı olarak küçültme. Bu nedenle, bu değişiklikler için katkıda bulunan etkenleri belirlemek için yeterli yağ dokusu toplama ve işleme gerekli4vardır.
Beyaz yağ dokular arasında subkutan ve viseral yağ depoları anatomik Lokalizasyon gibi farklı özelliklere sahip ve2,5işlev genel olarak kabul. Bu nedenle, çelişkili sonuçlar veya büyük değişkenlik önlemek için dikkat yağ yastıkları toplarken farklı bu yağ depoları arasında çapraz bulaşma önlemek için alınması gerekir.
Dahası, RNA veya protein fareler beyaz Yağ dokusundan izole zaman üç önemli sorunlar vardır. Farklı beyaz yağ depoları birbirinden ayıran sınır her zaman açık, diğer organlar böbrekler ve kalp6gibi aksine değildir gibi ilk olarak, obez farelerde yağ yastıkları toplama kolay bir iş değil. İkinci olarak, protein veya RNA izolasyon sırasında yağ dokusu yüksek lipid içeriği nedeniyle lipidler tabakası üst yüzen ve örnek için doğrudan erişimi engeller. Üçüncü olarak, kahverengi yağ dokusu veya diğer dokularda aksine, beyaz yağ dokusu önemli ölçüde daha düşük RNA ve protein içeriğe sahip ve bu genç fare kullanırken büyük endişe kaynağıdır, fareler bir normal (N) diyet beslenen ve için beklenen fareler var düşük yağ kütlesi (Yani KO modelleri, ilaçlar, tedavi egzersiz eğitim, vb.) 7 , 8.
Bu nedenle, RNA Yağ dokusundan izole etmek için uygun olan yöntemi seçimi önemlidir. Fenol/kloroform ayıklama için alternatif yöntemler ticari kitleri vardır. Onlar genellikle RNA arıtma tarafından takip sütun9tarihinde bir ilk fenol ayıklama adım temel alır. Ancak, bu kitleri normalde daha pahalı olduğunu ve RNA kalitesi değişken olabilir ise daha düşük verim, örnekleri vermek ama daha az zaman alıcı. Ancak, burada açıklanan fenol çözüm/kloroform çıkarma için en büyük avantajlarından biri RNA, DNA ve protein bir tek örnek10izole imkanı vardır. Çünkü fareler yağ yastıkları genellikle küçük ve RNA ve proteinler küçük miktar (özellikle yalın fare modellerinde) vermek, bu protokoller bir küçük bir örnek dışarı alabilirsiniz veri en üst düzeye çıkarmak.
Bu kağıt amacı üç fare beyaz yağ dokusu depoları hem de miktar ve RNA izolasyon kalitesi yeterli örnek koleksiyonundan emin olmak için bir yöntem ayrıntılı olarak tarif etmektir. Bu iletişim kuralı takip ederek elde RNA gerçek zamanlı PCR deneyleri gerçekleştirmek için kullanılabilir. Bu iletişim kuralı kültürlü birincil adiposit RNA izolasyonu için tasarlanmamıştır.

<table border="0" cellpadding="0" cellspacing="0" style="width:1003px;" width="1002">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">1 mL seringes</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1X TE solution (10 mM Tris-HCl and 1 mM EDTA&#8226;Na2. pH 8.0)</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">22 G needles</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">26 G needles</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">75% Ethanol</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Block heater (dry bath)</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Chloroform</td>
			<td>Sigma</td>
			<td>C2432-500mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">dATP&#160;&#160;&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">R0141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">dCTP&#160;&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">R0151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">dGTP&#160;&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">R0161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">DNase I (1 U/&#181;l)&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">EN0521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">dTTP&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">R0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Faststart Universal SYBR green Master (Rox)</td>
			<td style="width:189px;">Roche</td>
			<td style="width:189px;">4913922001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Faststart universal SYBR green master (Rox) fluorescent dye&#160;</td>
			<td style="width:189px;">Roche</td>
			<td style="width:189px;">4913914001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Filtered tips</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Forceps&#160;</td>
			<td style="width:189px;">Instrumentarium</td>
			<td>HB275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Gauze</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Hammer</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">High fat rodent diet</td>
			<td>Bio-Serv, Frenchtown, NJ</td>
			<td style="width:189px;">F3282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Isopropanol&#160;</td>
			<td>Laboratoire MAT</td>
			<td>IH-0101</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">Leptin forward PCR primer (5&#8217;-GGGCTTCACCCCATTCTGA-3&#8217;) 10 uM</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">Leptin reverse PCR primer (5&#8217;-GGCTATCTGCAGCACATTTTG-3&#8217;) 10 uM</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Liquid nitrogen</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Maxima Reverse Transcriptase (enzyme and 5x buffer)</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">EP0742</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Nanopure water (referred as ultrapure water)</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Nitrile examination gloves</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Nitrile gloves</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">Normal rodent diet</td>
			<td style="width:189px;">Harlan Laboratories, Madison, WI</td>
			<td style="width:189px;">Harlan 2018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">P1000 pipetman</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">P2 pipetman</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">P20 pipetman</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">P200 pipetman</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Phenol solution (TRIzol)&#160;</td>
			<td style="width:189px;">Ambion Life Technologies</td>
			<td style="width:189px;">15598018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Pre-identified aluminium foil</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quartz spectrophotometer cuvette</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rack for PCR tube strips</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Racks for RT-PCR tube strips</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Random hexamers&#160;</td>
			<td style="width:189px;">Invitrogen</td>
			<td style="width:189px;">58875</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:457px;">Real-time PCR Rotor Gene system&#160;</td>
			<td style="width:189px;">Corbett research</td>
			<td style="width:189px;">RG-3000 Rotor-Gene thermal cycler</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Refrigerated bench-top centrifuge</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RiboLock RNase Inhibitor&#160;</td>
			<td style="width:189px;">Thermo scientific</td>
			<td style="width:189px;">EO0381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RNase-free 1.5 mL eppendorf tubes</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RNase-free 1.5 mL screw cap tubes</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free PCR tube strips (0.2 mL) and caps</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RNase-free water&#160;</td>
			<td style="width:189px;">Hyclone</td>
			<td style="width:189px;">SH30538.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RT-PCR machine</td>
			<td style="width:189px;">Qiagen</td>
			<td style="width:189px;">Rotor-Gene Corbett 3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">RT-PCR tube strips (0.1 mL) and caps</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">S16 forward PCR primer (5&#8217;-ATCTCAAAGGCCCTGGTAGC-3&#8217;) 10 uM</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:457px;">S16 reverse PCR primer (5&#8217; ACAAAGGTAAACCCCGATCG-3&#8217;) 10 uM</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Spectrophotometer</td>
			<td style="width:189px;">Biochrom</td>
			<td style="width:189px;">Ultrospec 3100 pro</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Stainless steel mortar and pestle</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:457px;">Surgical pads</td>
			<td style="width:189px;">Home made a foam board wrapped in a disposable absorbent underpad</td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Surgical scissors&#160;</td>
			<td style="width:189px;">Intrumentarium</td>
			<td style="width:189px;">130.450.11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Thermal cycler</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Thermal cycler&#160;</td>
			<td style="width:189px;">Biometra</td>
			<td style="width:189px;">Thermocycler</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Vortex mixer</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Weighing spatula</td>
			<td style="width:189px;"></td>
			<td style="width:189px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse adipose tissue collection and processing for rna analysis

Çoğunlukla lipidler içerdiğinden diğer dokulara karşılaştırıldığında, beyaz yağ dokusu gibi gerçek zamanlı PCR ve Western Blot, karşıdan akış uygulamaları için bir önemli ölçüde daha az RNA ve protein içeriği vardır. Ek adımlar bu lipitlerin önlemek için gerektiğinde RNA izolasyon yağ dokusu örneklerinden de meydan okuyor. Burada, bu örnekler işlemek ve RNA izolasyon gerçekleştirmek için fare üç anatomik olarak farklı beyaz yağ dokuları toplamak için bir yordam mevcut. Biz daha fazla gerçek zamanlı PCR kullanarak cDNA ve gen ifade deneyleri sentezi tarif. Burada açıklanan protokol hayvanın saç ve kan yağ yastıkları yanı sıra doku toplama sırasında farklı yağ yastıkları arasında çapraz bulaşma kirlenme azalma sağlar. Bu da yeterli miktar ve çıkarılan RNA kalitesini sağlamak için optimize edilmiştir. Bu iletişim kuralı nerede yağ doku örnekleri gerçek zamanlı PCR gibi rutin deneyler için gerekli olan herhangi bir fare modeli için yaygın olarak uygulanabilir ama birincil adiposit hücre kültürü izolasyonu için uygun değildir.

Nekropsi yordamı izleyerek üç beyaz yağ dokuları toplanmış ve fareler (N ve HF diyet beslenen farelerin) iki grup ağırlıklı. Beklendiği gibi fareler HF diyet son vücut ağırlığı ve kilo alımı littermates N diyet (Tablo 1) göre artmıştı. Logosuna 2 kat artış PGF, PRF ve SCF ağırlığını obez farelerde N diyet'e kıyasla daha fazla bu gözlemler eşlik edildi.
İzole RNA ile herhangi bir deneyler gerçekleştirmeden önce saflığı 4,9 adımda anlatıldığı gibi değerlendirilmiştir. Her beyaz yağ dokusu için saf RNA (Tablo 2) olarak kabul edilir civarında 2.0 OD260/OD280 olduğunu oranı olarak örnekleri yeterli kalitede RNA izolasyon fenol çözüm tarafından üretilen. Gerçek zamanlı PCR veri leptin mRNA ifade PGF, PRF ve SCF obez farelerin N diyet (şekil 4A)'e göre önemli ölçüde arttı gösterdi. Nitekim, leptin mRNA ifadede gözlenen farklılıklar s16, Ct değerleri değil değiştirilmiş olarak fareler iki grup arasındaki sonuçlar normalleştirmek için kullanılan referans gen (şekil 4B) bir değişim nedeniyle değildi. HF diyet bir çalışma protokolü bir parametre belirtilir böylece, s16 güvenilir bir referans gen PGF, PRF ve SCF mRNA ifade için kullanılabilir.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57026/57026fig1.jpg"> Resim 1 . Farelerde beyaz yağ dokusunun anatomik Lokalizasyon. N diyet erkek fare her yağ dokusu depo ve böbrek üstü bezleri lokalizasyonu göstermek için davranışlarını kesip. SCF (A), (B)PGF, PRF (C) ve böbrek üstü bezi (D). PGF, peri Gonad yağ; PRF, peri-böbrek yağ; SCF, karın subcutaneous yağlı. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57026/57026fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57026/57026fig2.jpg"> Resim 2 . RNA izolasyon protokolü akış şeması kullanarak fenol çözüm. RNA topraklı beyaz Yağ dokusundan izole etmek için gereken adımları şematik gösterimi. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57026/57026fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57026/57026fig3.jpg"> Şekil 3 . Çamaşır suyu jel üzerinde RNA kalite değerlendirme. 1 ug subcutaneous yağlı (SCF) ve peri Gonad yağ (PGF) izole RNA'ın üzerinde % 1'özel beyazlatıcı Jel Elektroforez tarafından ayrılmış. 28s ve 18s rRNA UV-transillumination tarafından görüntülenmiştir. Jel SCF ve bir fare PGF izole RNA normal bir diyet beslenir. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57026/57026fig3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57026/57026fig4.jpg"> Şekil 4 . HF diyetin etkisi beyaz yağ dokusu leptin ve s16 mRNA ifadede. Leptin (A) ve s16 (B) mRNA ifade. Veri s16 için Ct değer olarak gösterilir ve her ikisi de ortalama ± SE n olarak sunulmaktadır leptin için veri s16 mRNA düzeyleri için normalleştirilmiş 12-13 grup başına =. * p < 0.05 N diyet için karşılaştırıldığında. N, normal; HF, yüksek yağ; PGF, peri Gonad yağ; PRF, peri-böbrek yağ; SCF, karın subcutaneous yağlı. Bu rakam Tan, değiştirilmiş olan P. ve ark, obezite (2014) 22, 2201-2209. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57026/57026fig4large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>N diyet</td>
			<td>HF diyet</td>
		</tr>
<tr>
<td>Son vücut ağırlığı (g)</td>
			<td>37,5 ± 1.3</td>
			<td>46,7 ± 1.7*</td>
		</tr>
<tr>
<td>Kilo alımı (g)</td>
			<td>6.6 ± 0.7</td>
			<td>16,7 ± 1.0*</td>
		</tr>
<tr>
<td>PGF (g)</td>
			<td>1,08 ± 0.17</td>
			<td>2,19 ± 0.15*</td>
		</tr>
<tr>
<td>PRF (g)</td>
			<td>0,79 ± 0,15</td>
			<td>1,71 ± 0.06*</td>
		</tr>
<tr>
<td>SCF (g)</td>
			<td>1.62 ± 0,35</td>
			<td>3,55 ± 0.20*</td>
		</tr>
</tbody></table>Tablo 1. Vücut ve beyaz yağ dokusu ağırlıkları HF diyet etkisi. Değerleri ifade aracı olarak ± SE n = 14-15 grup başına. * p < 0.05 N diyet için karşılaştırıldığında. N, normal; HF, yüksek yağ; PGF, peri Gonad yağ; PRF, peri-böbrek yağ; SCF, karın subcutaneous yağlı. Bu rakam Tan, değiştirilmiş olan P. ve ark, obezite (2014) 22, 2201-2209.
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>Örnekleri</td>
			<td>Oranı OD260/OD280</td>
		</tr>
<tr>
<td>PGF 1</td>
			<td>2.04</td>
		</tr>
<tr>
<td>PGF 2</td>
			<td>2.04</td>
		</tr>
<tr>
<td>PGF 3</td>
			<td>2,02</td>
		</tr>
<tr>
<td>DARBE TEKRARLAMA FREKANSI 1</td>
			<td>1,95</td>
		</tr>
<tr>
<td>DARBE TEKRARLAMA FREKANSI 2</td>
			<td>2.08</td>
		</tr>
<tr>
<td>DARBE TEKRARLAMA FREKANSI 3</td>
			<td>2.08</td>
		</tr>
<tr>
<td>SCF 1</td>
			<td>2.04</td>
		</tr>
<tr>
<td>SCF 2</td>
			<td>2,02</td>
		</tr>
<tr>
<td>SCF 3</td>
			<td>2.06</td>
		</tr>
</tbody></table>Tablo 2. RNA saflık beyaz yağ dokuları izolasyonu sonra. n = 3 yağ dokusu başına. PGF, peri Gonad yağ; PRF, peri-böbrek yağ; SCF, karın subcutaneous yağlı.

Watch this Scientific Journal Video about Mouse Adipose Tissue Collection and Processing for RNA Analysis at JoVE.com

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Fare yağ dokusu toplama ve işleme RNA analizi için

Bu kağıt amacı farklı beyaz yağ dokuları fareler toplamak, yağ örnekleri işlemek ve RNA ayıklamak için bir adım adım yordam sunmaktır.

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

mouse-adipose-tissue-collection-and-processing-for-rna-analysis

Nanyang Technological University

Research

JoVE Journal

Biochemistry

48.1K Görüntüleme.  Université de Montréal. Bu kağıt amacı farklı beyaz yağ dokuları fareler toplamak, yağ örnekleri işlemek ve RNA ayıklamak için bir adım adım yordam sunmaktır.

Video: Mouse Adipose Tissue Collection and Processing for RNA Analysis

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Lazer Diseksiyon RNA Sek için Deneysel Tasarım: Mısır Yaprak Kalkınma bir Analiz Dersler

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Biyokimya

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Kantitatif Transkriptom analizinde kullanım için tohumlar yüksek oranda saf RNA&#39;nın izolasyonu için etkili bir yöntem

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Çeviri başlatma karmaşık oluşumu memeli mRNA'ların Toeprinting analizi

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

Yakalama ve tıklama Kimya destekli RNA-interactome kullanarak RNA bağlanıcı proteinler tanımlaması (CARIC) strateji yakalama

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling

Fragrances of many flower families have been sampled and the volatiles analyzed. Knowing the compounds that make up the fragrances can be an important step to conservation of flowers that are threatened or endangered. Because floral fragrance is critical for attracting pollinators, this method could be used to better understand or even enhance pollination. We present a protocol using a portable charcoal air filter and vacuum to collect floral fragrance volatiles, which are then analyzed by a GC-MS. By using this method, fragrance volatiles can be sampled using a non-destructive method with a machine that is easily transported. This methodology uses a rapid sampling procedure, cutting sampling time down from 2-3 hours to approximately 10 minutes. Using GC-MS, the fragrance compounds can be identified individually, based on authentic standards. The steps used for collecting fragrance and control data are presented, from material setup to collecting the data output.

Here, we present a protocol for collecting the floral fragrance volatiles from blooming flowers, using a non-destructive sampling procedure.

GC-MS Termal Desorpsiyon Örneklemesi için Headspace Uçucu Toplama Tekniği kullanılarak Çiçek Kokusu Volatiles Hızlı Toplama

Fragrances of many flower families have been sampled and the volatiles analyzed. Knowing the compounds that make up the fragrances can be an important ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

RNA Interactors Protein kinaz memeli hücre döngüsü sırasında RNA-harekete geçirmek, eğitim

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

Rat Pankreas Dokusunun Hızlı ve Uygun Maliyetli RNA Ekstraksiyonu

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

Bitki MikroRNAlarının Saptanması ve Ölçülmesi için RNA Leke Analizi

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

Çeviri Yönetmeliğinde RNA-Protein Etkileşimlerini İncelemek için Optik Cımbız

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.