Bu eser tarafından desteklenmiştir fakülte araştırma hibe Hartford Üniversitesi, üniversite Sanatlar ve Bilimler Dekan'ın ofisinden.

Çalışma sırasında hayvan bakım ve tedavi Ulusal Sağlık Enstitüleri ilkesiyle uyulması, kurumsal kılavuzlarınıza uygun olarak vardı ve Hartford Üniversitesi hayvan kurumsal hayvan bakımı ve kullanımı Komitesi tarafından kabul edildi.
1. hayvanların sürüklenme
Not: 20 haftalık erkek B6129SF2/J fareler çalışmada kullanılır.
<ol><li>Fareler (Standart genel gider beyaz ışık) 12 h ışık için trene binmek / 12 h karanlık döngüsü 2 hafta önce deney için. Not: Burada zeitgeber zaman (ZT) 0 ışıkları ve ZT12, ışıkları tüm diğer çevresel faktörler (Örneğin, yiyecek, su ve oda sıcaklığında) tutarak karşılık gelen sabit 21.</li>
</ol>2. aletleri, kültür orta ve meydan okuma orta hazırlanması
<ol><li>Otoklav forseps ve diseksiyon makası. Buzlu mikroskop slaytlar alüminyum folyo ve basınçlı kap çiftlerini sarın.</li>
	<li>Kültür orta fetal Sığır serum (FBS) ekleyerek RPMI 1640 için % 10 son bir konsantrasyon için hazırlayın. Kültür ortamına aşağıdaki PAMPs ekleyerek 10 mL 50 mL tüpler ortamda meydan hazırlamak (RPMI % 10 ile FBS); LPS (5 µg/mL), ODN1826 (5 µg/mL), ısı-öldürdü Listeria Monositogenez (108 HKLM/mL), veya başka bir PAMP onun önerilen konsantrasyon.</li>
	<li>Sıcak kültür orta ve meydan okuma orta olarak 37 ° C'de bir su banyosu ve yaklaşık 70 mL steril fosfat tamponlu tuz (PBS, pH 7.2) Oda sıcaklığında tutmak.</li>
	<li>Kültür bir 10 mL pipet ve pipet yardım etmek için bir 50 mL tüp ve yer buz üzerinde kullanarak orta 10 mL ekleyin.</li>
	<li>Yaklaşık 30 mL % 70 etanol makas ve forseps mikrobiyal kontaminasyonu önlemek için kabı diseksiyon of a 100 mL kabı ve yer biter ekleyin.</li>
	<li>Lizis arabellek RNA izolasyon için 10 µL β-Mercaptoethanol (altında bir duman hood) ekleyerek arabellek RLT 50 mL tüp içinde her 1 mL hazır olun. Sadece gerekli (yaklaşık 1 x 106 hücreleri oluşur örnek başına 600 µL) olacak miktar olun. Not: Arabellek RLT silika membran için RNA bağlamayı destekler RNA ekstraksiyon kiti bir bileşenidir.</li>
</ol>3. Splenocyte yalıtım ve meydan okuma
<ol><li>Onları kendi özgün kafeste tutma ve 3 L/dak 1 dk. için CO2 durur nefes sonra tedarik devam akış hızında kafesine CO2 ekleyerek narkoz üzerinden belirli zeitgeber zaman fareler ötenazi.</li>
	<li>Servikal yerinden çıkması ile ölüm kafatası tabanında boynun iki tarafında başparmak ve işaret parmağı yerleştirerek onaylayın. Alternatif olarak, kafatası tabanında bir çubuk (Öte yandan kullanarak) hızlı bir şekilde kablolarıylaberaber kuyruk veya servikal vertebra ayrılması kafatası22neden arka bacaklarda Bankası basın.</li>
	<li>Fare gövde ile % 70 etanol ve kağıt havlu ile silin sprey. Üstünde onun sırt ve biraz eğik sağ yan üzerine fareyi getirin. Kürk kes gitsin, anatomi kullanarak, fare sol tarafında hakkında yarım ön ve arka bacakları makas.</li>
	<li>Forseps kullanarak, Karın zarını kapmak ve dikkatle dalak zarar için bir kesik yapın. Forseps ile dalak çıkarın ve Kültür medya buz üzerinde yaklaşık 10 mL içeren bir steril 50 mL tüp içine yerleştirin. Kalan hayvanlar için yineleyin. Not: Dalak bir börülce rengidir ve daha uzun ve böbrek daha düz.</li>
	<li>
Kültür Orta 2 mL ile bir dalak için küçük steril Petri kabına aktarın.
	<ol>
<li>Dalak iki steril buzlu slayt buzlu kısmı arasında taşlama tarafından lunaparkçı. Mümkünse, sorun ve hücreleri ortamda homojenizasyon işlemi sırasında tut.</li>
		<li>Bir kez iyice homojenize damlalıklı kültür splenocytes 50 mL tüp içine 40 µm naylon hücre süzgeç aracılığıyla içeren Orta 2 mL.</li>
		<li>Buzlu slaytları yeni çift, 50 mL tüpler kültür orta ve hücre Süzgeçler kullanarak kalan dalağı için yukarıdaki adımları yineleyin.</li>
	</ol>
</li>
	<li>Her splenocytes için toplam hacmi 10 mL/tüp içeren 50 mL tüpler 8 mL soğuk kültür ortamının ekleyin. Bir hemasitometre kullanarak mililitre başına hücre sayısını belirleyin.</li>
	<li>Yaklaşık 1 x 106 hücreler/iyi 6-şey kültür tabakaları bana ekleyin. Hücreleri de deney için kullanılan farklı PAMPs sayısına karşılık gelen ve bir denetim dahil kuyu sayısını ekleyin. 3 mL kültür ortamının veya meydan okuma orta 3 mL ilgili wells için ekleyin.</li>
	<li>Plakayı %5 CO2 3 h için 37 ° C'de kuluçkaya.</li>
	<li>Hücreleri de P1000 micropipette bağlı bir 1000 µL damlalıklı ucu kullanarak alt kazı. 15 mL tüp için aynı 1.000 µL damlalıklı ucu kullanarak hücreleri içeren orta aktarın.</li>
	<li>Santrifüjü 167 x g oda sıcaklığında 5 min için de üzerinden hücreleri cips. Süpernatant kaldırmak ve hücre Pelet 5 mL de PBS ile yıkayın.</li>
	<li>Hücreleri 167 x g de ikinci kez 5 min için cips, süpernatant kaldırın ve hücreleri parçalayıcı için hücre Pelet lizis arabelleği 600 µL ekleyin. Sonra RNA izolasyon için ilerlemek.</li>
</ol>4. RNA izolasyon ve cDNA sentezi
<ol><li>Splenocytes RNA ekstraksiyon kiti üreticinin yönergelerine uygun kullanarak gelen RNA yalıtmak ve 'isteğe bağlı' sütun üzerinde DNA sindirim gerçekleştirin.</li>
	<li>CDNA sentez önce RNA konsantrasyonu konsantrasyonu en uygun RNA aralığı (en çok 2 µg) içinde olduğunu doğrulamak için bir microvolume spektrofotometre kullanarak cDNA sentez Kit'in belirlemek.</li>
	<li>
Synthesize cDNA her üreticinin yönergelerine uygun ters transkripsiyon seti kullanarak örnekleri için. RNA'ın 10 µL her 20 µL toplam reaksiyon birimindeki örnekleri için kullanın. Ters transkripsiyon (thermocycler çalıştırmak) bitiminde 20 μL Ekle H2O her tepki için.
</li>
	<li>Standart eğri cDNA hazırlamak için 0.5 mL tüp içine birkaç denetim örneklerinden (Örneğin, 5 10 µL toplamda 2 örneklerinden µL) mRNA havuzu için bir P10 veya P20 micropipette kullanarak oluşturun.<ol>
<li>10 µL 2 x transkriptaz ana karışımı ekleyin.</li>
		<li>Ters transkripsiyon bitiminde, standart eğri seyreltme serisinde başlangıç konsantrasyonu ("1") görev yapacak tepki tüp H2O 10 µL ekleyin.</li>
		<li>10 kat seyreltme serisi (1-10-4) 45 µL P100 veya P200 micropipette 10-1, 10-2, 10-3ve 10-4dört 0.5 mL tüpler içine kullanarak su ekleyerek gerçekleştirin.<ol>
<li>5 µL P20 micropipettor, yukarı ve aşağı pipetting tarafından mix kullanarak başlangıç konsantrasyon "1" (10-1) ilk tüp içine eklemek birkaç kez, sonra transfer 5 µL tüp içine 10-1 tüp belirlenmiş 10-2 ve karışımı.</li>
			<li>5 µL P20 micropipette 10-2 tüp 10-3 özel tüp kullanarak transfer ve karıştırın. 5 µL P20 micropipettor 10-3 tüp 10-4 özel tüp kullanarak transfer ve karıştırın.</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. kantitatif polimeraz zincir reaksiyonu (qPCR)
<ol><li>MRNA düzeylerinin göreli Nefelometri qPCR tarafından belirler. İçinde deneysel Kur'u "Nefelometri - göreli standart eğri" ve "TaqMan" Kimya seçin. Değişiklik tepki birime 10 µL. plaka düzenine (Örneğin, hedef gen, standart eğri, muhabir, vb) ilgili bilgileri girin.</li>
	<li>İçerdiği 0.5 µL astar/sonda testin, gen ifade ana karışımı 5 µL, H2O 2 µL ve 2.5 tepki hazırlıkları µL cDNA (10-100 ng).<ol>
<li>Bu standart eğri eklediğinizden emin reaksiyonlar, negatif denetimleri, her tepki performans yinelenen ve hesap olacaktır 2 İlave reaksiyonlar sayısına göre her biri önceki reaktifleri (hariç cDNA) çarparak ana bir karışım hazırlamak Pipetting değişim için.</li>
		<li>Damlalıklı 7.5 µL bir 96-iyi tepki plaka bir micropipette veya bir çok kanallı pipet ile önceden belirlenmiş kuyu içine Ana hazırlanan karışımı. Damlalıklı cDNA bilinmeyenli ve standartları için uygun kuyunun içine 2.5 µL ve H2O negatif kontrol kuyu içine 2.5 µL.</li>
		<li>Çeşitli moleküler saat genler için astar/sonda deneyleri ve aynı zamanda bir tahlil için bir endojen denetimi (Örneğin, β-aktin) içerir.</li>
	</ol>
</li>
	<li>Göreli ifade değerleri belirlemek için her çoğaltma için Ortalama göreli miktarını hesaplamak sonra her örnek için hedef gen için Ortalama göreli miktarı ile β-aktiniçin Ortalama göreli miktarı bölün.</li>
</ol>6. istatistiksel analiz
<ol><li>İstatistiksel analiz yazılımı kullanarak, veri sütun istatistikleri seçeneği altında programa girin. Bir tek yönlü ANOVA saat Gen ifadesinin denetimi karşı PAMP meydan sonra ortalama değerleri arasındaki farklar değerlendirmek için Dunnett'ın öğleden hoc testi ile seçin.</li>
</ol>

<ol>
	<li>Bell-Pedersen, D., 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=Circadian+rhythms+from+multiple+oscillators:+lessons+from+diverse+organisms.">Circadian rhythms from multiple oscillators: lessons from diverse organisms.</a> Nat. Rev. Genet. 6, 544-556 (2005).</li><li>Mohawk, J. A., Green, C. B., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+and+Peripheral+Circadian+Clocks+in+Mammals.">Central and Peripheral Circadian Clocks in Mammals.</a> Annu. Rev. Neurosci. 35, 445-462 (2012).</li><li>Balsalobre, A., Damiola, F., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serum+shock+induces+circadian+gene+expression+in+mammalian+tissue+culture+cells.">A serum shock induces circadian gene expression in mammalian tissue culture cells.</a> Cell. 93, 929-937 (1998).</li><li>Yoo, S. -. H., 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=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc. Natl. Acad. Sci. U. S. A. 101, 5339-5346 (2004).</li><li>Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+Central+and+Peripheral+Circadian+Oscillators+in+Transgenic+Rats.">Resetting Central and Peripheral Circadian Oscillators in Transgenic Rats.</a> Science. 288, 682-685 (2000).</li><li>Lowrey, P. L., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+circadian+rhythms+in+mammalian+model+organisms.">Genetics of circadian rhythms in mammalian model organisms.</a> Adv. Genet. 74, 175-230 (2011).</li><li>Curtis, A. M., Bellet, M. M., Sassone-Corsi, P., O'Neill, L. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+Clock+Proteins+and+Immunity.">Circadian Clock Proteins and Immunity.</a> Immunity. 40, 178-186 (2014).</li><li>Keller, M., 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=A+circadian+clock+in+macrophages+controls+inflammatory+immune+responses.">A circadian clock in macrophages controls inflammatory immune responses.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 21407-21412 (2009).</li><li>Silver, A. C., Arjona, A., Hughes, M. E., Nitabach, M. N., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+expression+of+clock+genes+in+mouse+macrophages,+dendritic+cells,+and+B+cells.">Circadian expression of clock genes in mouse macrophages, dendritic cells, and B cells.</a> Brain. Behav. Immun. 26, 407-413 (2012).</li><li>Kawai, T., Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pattern-recognition+receptors+in+innate+immunity+update+on+Toll-like+receptors.">The role of pattern-recognition receptors in innate immunity update on Toll-like receptors.</a> Nat. Publ. Gr. 11, 373-384 (2010).</li><li>Marpeg&#225;n, L., Bekinschtein, T. A., Costas, M. A., Golombek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+responses+to+endotoxin+treatment+in+mice.">Circadian responses to endotoxin treatment in mice.</a> J. Neuroimmunol. 160, 102-109 (2005).</li><li>Okada, K., 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=Injection+of+LPS+Causes+Transient+Suppression+of+Biological+Clock+Genes+in+Rats.">Injection of LPS Causes Transient Suppression of Biological Clock Genes in Rats.</a> J. Surg. Res. 145, 5-12 (2008).</li><li>Haimovich, B., 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=In+vivo+endotoxin+synchronizes+and+suppresses+clock+gene+expression+in+human+peripheral+blood+leukocytes.">In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes.</a> Crit. Care Med. 38, 751-758 (2010).</li><li>Curtis, A. M., 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=Circadian+control+of+innate+immunity+in+macrophages+by+miR-155+targeting+Bmal1.">Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1.</a> Proc. Natl. Acad. Sci. U. S. A. 112, 7231-7236 (2015).</li><li>Silver, A. C., Arjona, A., Walker, W. E., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Circadian+Clock+Controls+Toll-like+Receptor+9-Mediated+Innate+and+Adaptive+Immunity.">The Circadian Clock Controls Toll-like Receptor 9-Mediated Innate and Adaptive Immunity.</a> Immunity. , (2012).</li><li>Gibbs, J. E., 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+nuclear+receptor+REV-ERB+&#945;+mediates+circadian+regulation+of+innate+immunity+through+selective+regulation+of+inflammatory+cytokines.">The nuclear receptor REV-ERB &#945; mediates circadian regulation of innate immunity through selective regulation of inflammatory cytokines.</a> PNAS. 109, 582-587 (2012).</li><li>Zee, P. C., Attarian, H., Videnovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+abnormalities.">Circadian rhythm abnormalities.</a> Contin. Lifelong Learn. Neurol. 19, 132-147 (2013).</li><li>Bovbjerg, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+disruption+and+cancer:+sleep+and+immune+regulation.">Circadian disruption and cancer: sleep and immune regulation.</a> Brain. Behav. Immun. 17, 48-50 (2003).</li><li>Fu, L., Lee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock:+pacemaker+and+tumour+suppressor.">The circadian clock: pacemaker and tumour suppressor.</a> Nat. Rev. Cancer. 3, 350-361 (2003).</li><li>Germain, A., Kupfer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIRCADIAN+RHYTHM+DISTURBANCES+IN+DEPRESSION.">CIRCADIAN RHYTHM DISTURBANCES IN DEPRESSION.</a> Hum. Psychopharmacol. 23, 571-585 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Mouse+Care+and+Maintenance.">Basic Mouse Care and Maintenance.</a> JoVE. , (2018).</li><li>Leary, S., 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=AVMA+Guidelines+for+the+Euthanasia+of+Animals+:+2013+Edition.">AVMA Guidelines for the Euthanasia of Animals : 2013 Edition.</a> AVMA. , (2013).</li><li>Silver, 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=Pathogen-associated+molecular+patterns+alter+molecular+clock+gene+expression+in+mouse+splenocytes.">Pathogen-associated molecular patterns alter molecular clock gene expression in mouse splenocytes.</a> PLoS One. , 12-15 (2017).</li><li>Silver, A. C., 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=Daily+oscillations+in+expression+and+responsiveness+of+Toll-like+receptors+in+splenic+immune+cells.">Daily oscillations in expression and responsiveness of Toll-like receptors in splenic immune cells.</a> Heliyon. , 00579 (2018).</li></ol>

Yazar ifşa etmek hiçbir şey vardır.

Bu protokolde, microvolume spektrofotometre ölçmek ve gen ekspresyonu belirlemede kullanılan RNA saflığı değerlendirmek için kullanılabilir. Nükleik asitler absorbe UV ışığı 260 nm, proteinler genellikle 280 ışığı absorbe bir RNA çıkarma işlemi (Örneğin, fenol) sırasında kullanılan diğer potansiyel kirletici 230 tespit olmakla birlikte nm, nm. Bu nedenle, absorbans (bir) oranında 260/280 değerlendirme tarafından nm (RNA protein) ve 260/230 nm (protein olmayan kirletici için RNA) ...

Neredeyse tüm yönleri için 24 h salınımlarını fizyolojisi ve davranış orchestrates, ana saat memelilerde, suprachiasmatic çekirdek hipotalamus1,2içinde (SCN) yer alır. Bir organizma düzeyinde biyolojik süreçleri düzenleyen ek olarak, ana saatini de periferik hücresel saatler boyunca vücut3,4,5eşitler. En az üç birbirine translasyonel transkripsiyon geri besleme döngüleri moleküler saat makine oluşur ise, çekirdek dönemi (Per1-3), Cryptochrome (Cry1-2), Bmal1oluşur, ve saat genler6,7. Çekirdek moleküler saat doğru zamanlama sürdürmek, yanı sıra bazı yardımcı saat gen ürünleri (Örneğin, Rev erbα ve Dbp) da ifade olmayan genlerine düzenleyen, Yani, kontrollü genler (CCGs)6 saat , 7.
Fonksiyonel moleküler saatler çeşitli bağışıklık dokuları (Örneğin, dalak ve lenf bezleri)8 ve hücreleri (Örneğin, B hücreleri, dendritik hücreler, makrofajlar)8,9tarif edilmistir. Bu hücreler algılamak ve moleküler desenleri patojen ilişkili (PAMPs), doğuştan gelen bağışıklık tanıma reseptörleri Toll benzeri reseptörler (TLR)10gibi ile korunmuş mikrobiyal bileşenleri yanıt. Bugüne kadar 13 fonksiyonel TLRs, hangi bakteri hücre duvarı bileşenleri, kamçılı protein ve mikrobiyal nükleik asitler10gibi mikrobiyal bileşenleri tanımak tarif edilmistir. PAMP, lipopolysaccharide (LPS), gram-negatif bakteri TLR4 tarafından tanınan bir hücre duvarı bileşeni sirkadiyen ritim, organizma ve moleküler düzeyde değiştirmek için gösterilmiştir. Örneğin, LPS vivo içinde meydan etkinlik fareler11 tarafından ölçülen fotosentez benzeri faz gecikmeler indüklenen ve indirimli saat gen ekspresyonu SCN (sahne) ve in situ hibridizasyon ve kantitatif PCR ile belirlendiği gibi karaciğer yol açtı, sırasıyla, içinde12sıçanlar. Sonra bir vivo içinde sorun LPS ile qPCR ile ölçülen birkaç saat genlerin değişmiş ifade insan periferik kan lökosit13 ve deri altı yağ dokusu14 analizi ortaya koydu. Son olarak, insan makrofajlar ve fare peritoneal makrofajlar, ayrıca yol açtı ex vivo LPS sorunları qPCR14tarafından ölçülen saat ifade değiştirdi.
Burada, biz PAMPs LPS, (sentetik oligonucleotides içeren bazlar CpG motifler) ODN1826, etkisini değerlendirmek için bir protokol tanımlamak ve TLR4, TLR9 ve TLR2, tarafından tanınan ısı öldürdü Listeria Monositogenez (HKLM), sırasıyla, Moleküler saat Gen ifadesinin içinde fare splenocytes. Protokol fare splenektomi, splenocyte yalıtım ve meydan okuma, RNA ayıklama, cDNA sentezi ve qPCR birkaç saat genlerin ifade değerlendirmek için içerir. Bu iletişim kuralı çok az hayvan ile bağışıklık hücreleri çok sayıda zamanında edinimi için izin verir veya daha sonra olabilir hücresel manipülasyon ex vivo çeşitli PAMPs ile meydan. Moleküler saat bağışıklık yanıtı8,15,16, çeşitli açılardan modüle gösterilmiştir, bu nedenle, moleküler saat çalışmasının büyük olasılıkla uygun zamana bağımlı varyasyonu zarar bağışıklık yanıtı. Sirkadiyen ritim kesintileri ciddi patolojiler17,18,19,20' ye neden olabilir beri Ayrıca, araştırmacılar splenocytes geniş bir yelpazede meydan okumak için ilgi olabilir moleküller ve saat onların etkisi değerlendirmek.

<table border="0" cellpadding="0" cellspacing="0" style="width:796px;" width="795">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Frosted slides</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:137px;">12-550-343</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell strainers</td>
			<td>Fisher</td>
			<td align="right">22363547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipopolysaccharide&#160;</td>
			<td>InvivoGen</td>
			<td>ltrl-eklps</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ODN1826</td>
			<td>InvivoGen</td>
			<td>Tlrl-1826-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HKLM</td>
			<td>InvivoGen</td>
			<td>Tlrl-hklm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td>20012-043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104 or 74106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td align="right">79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well cell culture plate</td>
			<td>Denville</td>
			<td>T1006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tubes</td>
			<td>Corning</td>
			<td align="right">352070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml tubes</td>
			<td>Corning</td>
			<td align="right">352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher</td>
			<td align="right">4368814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays b-actin</td>
			<td>ThermoFisher</td>
			<td>Mm00607939_s1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Per2</td>
			<td>ThermoFisher</td>
			<td>Mm00478113_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Rev-erba</td>
			<td>ThermoFisher</td>
			<td>Mm00520708_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Bmal1</td>
			<td>ThermoFisher</td>
			<td>Mm00500226_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Dbp</td>
			<td>ThermoFisher</td>
			<td>Mm00497539_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR machine StepOnePlus</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>ThermoFisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Fast 96-well reaction plate (0.1 ml)</td>
			<td>ThermoFisher</td>
			<td align="right">4346907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Statistical Analysis Software</td>
			<td>Prism 7.0a</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

Davranış-gen ekspresyonu sirkadiyen ritim fizyolojisi neredeyse tüm yönlerini düzenleyen. Burada, fare splenocytes moleküler desenleri (PAMPs) patojen ilişkili lipopolysaccharide (LPS), ODN1826 ve ısı öldürdü Listeria Monositogenez meydan ve moleküler üzerindeki etkileri sirkadiyen incelemek için bir metodoloji mevcut saat. Daha önce çalışmalar LPS etkisi a değişiklik-in vivo ve ex vivo yaklaşımlardan modelleri (Örneğin, fare, sıçan ve insan) bir ürün yelpazesine kullanarak moleküler saat inceleyerek üzerinde odaklanmıştır. Bu iletişim kuralı yalıtım ve saat gen ifade sonrası meydan kantitatif PCR ile değerlendirmek için meydan okuma splenocytes yanı sıra metodoloji açıklanmaktadır. Bu yaklaşım sadece etkisi mikrobiyal bileşenleri ifade saatin değiştirebilir moleküler saat diğer moleküller de ama değerlendirmek için kullanılabilir. Bu yaklaşım ayrı nasıl etkilediği PAMP Toll benzeri reseptör etkileşim saat ifade moleküler mekanizması kızdırmak için yararlanılabilir.

Fareler ZT13 kurban edildi, splenocytes izole edildi ve ex vivo PAMPs LPS, ODN1826 veya HKLM meydan. 3 h sonra RNA izole edildi ve qPCR saat, Per2, Dbp ve Rev-erbα tartışmasız denetim hücrelere kıyasla moleküler saat genlerin göreli ifade düzeylerini değerlendirmek için kullanılır. PAMP meydan sonra saat ifade düzeyleri kontrol hücreleri (Şekil 1A) ifadesinde önemli ölç?...

Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Bu protokolü moleküler saat gen ekspresyonu alter patojen ilişkili moleküler desenleri bulmak için fare splenocytes kullanarak bir teknik anlatılmaktadır.

Saat Gen ifadesinin etkisi moleküler desen patojen ilişkili değerlendirmek için fare Splenocytes kullanımı

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

the-use-mouse-splenocytes-to-assess-pathogen-associated-molecular

Research

JoVE Journal

Genetics

7.4K Görüntüleme.  University of Hartford. Bu protokolü moleküler saat gen ekspresyonu alter patojen ilişkili moleküler desenleri bulmak için fare splenocytes kullanarak bir teknik anlatılmaktadır.

Video: The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

İnsan Primer Hücre Kültürleri içinde Sirkadyen Saat Gen İfade ve Hormon salgısını Paralel Ölçümü

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Genetik

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Multiplex RT-qPCR kullanarak tek hücreli Gen İfadesi Nadir Lenfoid Populasyonlarının Farklılıkları karakterize etmek

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

Hücre Çevrimiyle Düzenlenen Gen İfadesini İki Tamamlayıcı Hücre Senkronizasyon Protokolü İle Çalışmak

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

Numune hazırlama ve analiz RNASeq tabanlı gen ifadesi verileri Zebra balığı

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Yerinde mutagenisitesinin CRISPR/Cas9 ve SsODN insan hücrelerinde tarafından katalize nokta mutasyon onarım bir fonksiyonu olarak incelemek için standart bir metodoloji

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Üretim ve Baculovirus arınma gen tedavisi uygulaması için

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Bir Optogenetic kontrol etmek ve hücre-hücre etkileşimleri gen ifade desenleri analiz yöntemi

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

Thromboxane A2 reseptör gen C924T polimorfizmi çalışmak için bir yöntem

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Mayoz sırasında insan gen işlevini değerlendirmek için fare yumurta kullanarak ben

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

Kök Microbiome keşfetmek: toprak, rizosferde ve kök Endosphere bakteriyel topluluk verileri ayıklama

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...