C.Y.W. received support from the National Health Research Institute (NHRI-EX101-9925SC), the National Science Council (101-2314-B-182-100-MY3, 101-2314-B-182A-009), and Chang Gung Memorial Hospital (CMRPG3B1643, CMRPG3D1002, CMRPG3D0581, CMRPG380091, and CMRPG3C1763).

NOT: 6 A level rRNA, mRNA ve diğer küçük RNA&#39;lar dahil toplam RNA m. Ribozomal RNA bol M 6 A değişiklikleri, 6 A seviyesi bu gerçeği dikkate almak gerekir m ölçümünü beri. 1. RNA İzolasyon <ol><li> (Kağıt havlu üzerine püskürtülen) ribonükleaz dekontaminasyon solüsyonu kullanarak, pipet ve laboratuar tezgah yüzeyleri silin. Islak kağıt nükleaz içermeyen su ile havlu ve tekrar pipet ve laboratuvar tezgah yüzeyleri silin. </li><li> RNA Ekstraksiyonu <ol><li> Toplam RNA İzolasyon <ol><li> Bir kafa üstü karıştırıcı kullanılarak, doku, 50-100 mg ya da 4 ° C &#39;de muhafaza RNA izolasyon çözeltisi 1 mL 5-10 x 10 6 hücre homojenleştirin. Not: fazla doku (100-150 mg), çünkü burada daha düşük RNA konsantrasyonların farelerden adipoz doku örnekleri için gerekli olabilir. Örnekler fare kalp, karaciğer, iskelet kası, akciğer, beyin kaynaklı ve makrofajlar, daha önce 4 kullanılmıştır. </li><li> İçindeOda sıcaklığında 5 dakika boyunca numune homojenatları cubate. </li><li> Örnek homojenat 1 ml başına 1-bromo-3-kloropropan (BCP) 100 mcL ekleyin. 15 saniye boyunca kuvvetli bir şekilde çalkalanır tüpleri ve üretici talimatlarına 24&#39;e göre, toplam RNA izole etmek için devam edin. </li></ol></li><li> İzole edilmiş toplam RNA&#39;dan poliadenile mRNA Saflaştırma <ol><li> olarak temin edilebilen kitler veya protokoller kullanılarak poliadenile edilmiş mRNA&#39;nın arındırın. <ol><li> Aşağıdaki çözeltinin her yeniden askıya iyice çalkalanır: 2x bağlama çözeltisi, oligo (dT) polistiren boncuklar ve yıkama solüsyonu. oligo (dT) kullanılmadan önce oda sıcaklığına getirin boncuk emin olun. </li><li> bir ısıtma bloğu içinde 70 ° C&#39;de bir boru ve ısıya hazırlık başına elüsyon çözeltisi 120 uL aktarın. </li><li> Bir mikrosantrifüj tüpe toplam RNA 500 mg kadar pipetle. nükleaz içermeyen su ile 250 mcL ses seviyesini ayarlamak. </li><li> toplam RNA 2x bağlayıcı çözeltisi 250 uL ekleyin böylecedökülmesinden ve vorteks kısaca içeriğini karıştırmak için. </li><li> oligo 15 mcL ekleyin (dT) boncuk ve vorteks iyice karıştırın. </li><li> 3 dakika boyunca 70 ° C&#39;de inkübe karışımı. </li><li> Isıtma bloğu numuneyi çıkarın ve 10 dakika boyunca oda sıcaklığında bekletilir. </li><li> 2 dakika için 15,000 x g&#39;de santrifüjleyin. </li><li> Dikkatle arkasında yaklaşık 50 uL bırakarak süpernatant kaldırmak. </li><li> pipetleme pelet yeniden askıya yıkama solüsyonu 500 mcL ekleyin. </li><li> Bir spin filtre / toplama tüpü kümesine süspansiyonu pipetle. </li><li> 2 dakika için 15,000 x g&#39;de santrifüjleyin. toplama tüpünden sütunu kaldırmak ve flow-through atın. toplama tüpüne sütun dönün. </li><li> Spin filtre üzerine yıkama solüsyonu Pipet 500 uL. </li><li> 15.000 x santrifüj 2 dakika boyunca örn. </li><li> taze toplama tüpüne sıkma filtre aktarın. </li><li> Elüsyon çözeltisi pipetle 50 uL ısıtıldı70 ° C&#39;ye kadar eğirme filtrenin merkezi üzerine. </li><li> 70 ° C&#39;de 2-5 dakika boyunca inkübe edin. 1 dakika için 15,000 x g&#39;de santrifüjleyin. </li><li> Spin filtrenin merkezi üzerine 70 ° C&#39;ye kadar ısıtıldı elüsyon çözeltisi ilave 50 uL pipetle. </li><li> Tekrarlayın Adım 1.2.2.1.18. </li></ol></li><li> 100 ug / mL glikojen, 3 M sodyum asetat tamponu (pH 5.2), ve mutlak etanolün 2.5 hacim 0.1 hacim. -80 ° C de bir gece hızlandırabilir. </li><li> 4 ° C&#39;de 25 dakika boyunca 15,000 x g&#39;de santrifüjleyin. süpernatant atın. </li><li> % 75 etanol içindeki 1 ml pelet yıkayın. 4 ° C&#39;de 15 dakika boyunca 15,000 x g&#39;de santrifüjleyin. Dikkatlice etanol çıkarın. </li><li> 3-5 dakika boyunca hava içinde kurutulur. </li><li> pelet çözmek için nükleaz içermeyen su 10 uL ekleyin. </li><li> -70 ° C&#39;de saklayın. </li></ol></li></ol></li><li> RNA Yeterlilik ve miktarının belirlenmesi <ol><li> Bir spektrofotometre kullanılarak, Notl RNA konsantrasyonunu belirlemek260 nm ve 280 nm &#39;de absorbans ng. A 260 / A 280 oranı 1.8-2.2 olmalıdır. </li><li> % 0.8 agaroz jel elektroforezi 25 kullanılarak RNA örnek kalitesini kontrol edin. NOT: sağlam ökaryotik toplam RNA yoğun 28S ve 18S rRNA bantları göstermesi gerekir. İyi bir RNA preparasyonu için 18S rRNA bandı şiddeti ile 28S oranı ~ 2. </li></ol></li></ol> 2. Jel Elektroforez ve Transferi NOT: Tampon hazırlama protokolleri Tablo 1&#39;de verilmiştir. <ol><li> Formaldehid jel hazırlanması (% 1) <ol><li> diethylpyrocarbonate (DEPC) su ile tüm elektroforez cihazları durulayın. </li><li> Tamamen bir mikrodalga fırınında DEPC su 215 mL agaroz 2.5 g eritin. </li><li> 10x 3- (N-morfolino) propansülfonik asit (MOPS), 12.5 ml tampon ve% 37 formaldehid 22.5 ml. </li><li> 1.5 mm kalınlığında tarak ile elektroforez cihazının içine dökün. </li><li> t kabarcıkları ortadan kaldırmak veya itmekTemiz bir tarak ile jel kenarlarına hem. </li><li> Jel, oda sıcaklığında katılaşmaya izin verin. </li></ol></li><li> Örnek hazırlama <ol><li> örnek tamponu toplam RNA 1-10 ug ve 11.3 uL karıştırın. </li><li> örnek tampon 11.3 uL RNA işaretleyici (1 mcg / ml) 2 ug karıştırın. </li><li> 16 uL bir toplam hacimde 2.2.1 ve 2.2.2 örnekler nükleaz içermeyen su ilave edilir. </li><li> 5 dakika boyunca 60 ° C de ısı ve daha sonra buzda. </li><li> buz üzerinde / ul etidyum bromür 0.1 ug içeren izleme boya 4 uL ile 2.2.3 den örnek 16 mcL karıştırın. DİKKAT: Solunduğunda Ethidium bromür muhtemelen teratojenik ve toksiktir. etidyum bromür güvenlik formu okuyun. </li></ol></li><li> Elektroforez <ol><li> 10x MOPS 200 mL 1x MOPS çalışan tampon yapmak için GKD 2 O 1.800 ml tampon ekleyin. </li><li> jel kuyu durulayın 1x MOPS çalışan tampon kullanın. </li><li> Ön-run tO 5 dakika boyunca 20 V jel. </li><li> Jelin kuyuların içine RNA örnekleri yükleyin. </li><li> Işık maruz kalmasını önlemek için folyo ile örtün. (Gece ​​boyunca) yaklaşık 17 saat süre ile 35 V çalıştır </li><li> Incelemek ve bir UV ışığı altında jel fotoğrafını. </li></ol></li><li> transfer <ol><li> Kullanılmayan kısmı kaldırmak için jel kesti. </li><li> 10x SSC tamponu (20x SSC tamponu 250 mL DEPC su 250 mL) 500 mL hazırlayın. </li><li> 50 rpm&#39;de sallama ile 20 dakika için 10 x SSC tamponu ile iki kere jel yıkayın. </li><li> Transfer tepsi (Şekil 1) transfer tamponu emen bir fitil kağıt, olarak hizmet verecek kadar büyük 1 filtre kağıdı sayfasını kesin. </li><li> jel aynı boyutta filtre kağıdı 4 parça kesin. </li><li> jel aynı boyuta pozitif yüklü bir naylon zar 1 sayfa kesin. </li><li> jel üzerinde çentik ve, uygun yönlenmesinin sağlanması için bir işaretleyici olarak membran işaretleyin. </li><li> 15 dakika boyunca 2 x SSC tamponu membran bekletin. </li><li> 500 ml o dökünf 10x SSC transferi tepsisine tampon. </li><li> cam levha üzerinde büyük filtre kağıdı sayfasını Lay ve transfer tamponu ile filtre kağıdı ıslatın. </li><li> Bir pipet yardımıyla kabarcıkları dışarı rulo. </li><li> transfer tamponuna önceden kesilmiş filtre kağıdı 2 adet ıslatın ve adım 2.4.5 filtre kağıdının ortasına koyun. herhangi bir kabarcıklarını çıkarmak. </li><li> aşağı filtre kağıdı üzerinde jel üst yan yattı. </li><li> jel üstünde membran yerleştirin. jel ve membran çentikler aynı hizaya getirin. </li><li> membran üstüne önceden kesilmiş filtre kağıdı 2 adet koyun ve transfer tamponu ile filtre kağıdı ıslatın. </li><li> tabakalar arasındaki herhangi bir baloncukları giderin. </li><li> havlu sadece jel ve zarından geçen tampon kadar emmek emin olmak için jel etrafında plastik wrap uygulayın. </li><li> Filtre kağıdı üzerine kağıt havlu Stack. </li><li> havlunun üstünde bir büyük boy cam plaka koyun. </li><li> yığının üstüne bir ağırlık yerleştirin. </li><li>RNA membrana jelden transfer etmek için gece boyunca devam edin. </li></ol></li></ol> N 6 -methyladenosine 3. Algılama <ol><li> membran Çapraz bağlama <ol><li> 2x SSC tamponu nemli membran tutun. </li><li> UV çapraz bağlayıcı içine 10x SSC tamponu ile dalmış filtre kağıdı 2 yaprak yerleştirin. </li><li> RNA adsorbe tarafı yukarı bakacak şekilde, filtre kağıdının üstüne membran yerleştirin. </li><li> &quot;Autocrosslink modu&quot; (120,000 μJ) seçin ve ışınlama başlatmak için &quot;Başlat&quot; düğmesine basın. </li><li> membrana jelden RNA transferi onaylamak için UV jel görüntü alıcı ile membran ve jel inceleyin. </li></ol></li><li> Bağışıklık <ol><li> 1 saat boyunca oda sıcaklığında Tween 20 (TBST) tampon Tris-tamponlu tuzlu su içinde% 5 yağsız süt içinde membran engeller. </li><li> 15 dakika boyunca TBST tamponu ile üç kez yıkayın. </li><li> Gecede m 6 A (membran inkübe &lt;em&gt; K 6 -methyladenosine) antikor çözeltisi (1: 1000,% 5 yağsız süt TBST tamponu) 4 ° C&#39;de ilave edildi. </li><li> 15 dakika boyunca TBST tamponu ile üç kez membran yıkayın. </li><li> Oda sıcaklığında 1 saat boyunca (% 5 yağsız süt TBST tamponu içinde 2.000: 1) HRP ile konjüge edilmiş eşek anti-tavşan antikoru çözelti içinde membran inkübe edin. </li><li> 15 dakika boyunca TBST tamponu ile üç kez membran yıkayın. </li><li> Gelişmiş kemiluminesans substrat (membran cm2 başına 0.125 ml) uygulayın. </li><li> Imalatçının talimatlarına 26 göre bir dijital kamera optimum ayarlarla kemilüminesans yakalar. </li></ol></li><li> m 6 A Niceleme <ol><li> ImageJ yazılımı kullanarak göreceli m 6 A kemilüminesan yoğunluğunu ölçmek. <ol><li> ImageJ yazılım menüsü altında ilgili dosyayı açmak için &quot;Dosya&quot; seçeneğini seçin. </li><li> ImageJ gelen &quot;Dikdörtgen&quot; aracını seçin ve etrafında bir çerçeve çizinsinyaller. </li><li> Ribozomal RNA deneyleri odağı değilse, hesaplama için 18S ve 28S ribozomal RNA m 6 A bantları kaçının. </li><li> Seçilen şeritli onaylamak için &quot;Command&quot; ve &quot;1&quot; i tıklayın. </li><li> Basın &quot;Command&quot; ve &quot;3&quot; Seçilen komplo göstermek için. </li><li> &quot;Düz&quot; aracını tıklayın ve parçalı alanı ayırmak için çizgiler çizmek. </li><li> ölçümleri kaydetmek için &quot;Wand&quot; aracını tıklayın. </li><li> veri verir. </li><li> Etidyum bromür boyama gelen 18S ribozomal RNA bant ile m 6 A düzeylerini normale. </li></ol></li></ol></li></ol>

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Y., 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=FTO+modulates+fibrogenic+responses+in+obstructive+nephropathy.">FTO modulates fibrogenic responses in obstructive nephropathy.</a> Sci Rep. 6, 18874 (2016).</li><li>Li, X., 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=Transcriptome-wide+mapping+reveals+reversible+and+dynamic+N-methyladenosine+methylome.">Transcriptome-wide mapping reveals reversible and dynamic N-methyladenosine methylome.</a> Nat Chem Biol. , (2016).</li><li>Sanschagrin, S., Yergeau, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+of+16S+ribosomal+RNA+gene+amplicons.">Next-generation sequencing of 16S ribosomal RNA gene amplicons.</a> J Vis Exp. (90), (2014).</li><li>Kukutla, P., Steritz, M., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+ribosomal+RNA+for+mosquito+gut+metagenomic+RNA-seq.">Depletion of ribosomal RNA for mosquito gut metagenomic RNA-seq.</a> J Vis Exp. (74), (2013).</li><li>Mishima, 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=Immuno-Northern+Blotting:+Detection+of+RNA+Modifications+by+Using+Antibodies+against+Modified+Nucleosides.">Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.</a> PLoS One. 10 (11), 0143756 (2015).</li><li>Klungland, A., Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+RNA+modifications+in+disease.">Dynamic RNA modifications in disease.</a> Curr Opin Genet Dev. 26, 47-52 (2014).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Modifications of RNA have important roles in cellular function and physiology. The current understanding of the regulation, function, and homeostasis of these modifications is still being explored and expanded8. Therefore, a precise and gold-standard method to evaluate the modifications of RNA is needed. The modified northern blotting method provides precise quantification of RNA modifications and clear delineation of the modifications in diverse RNAs. Although the method requires at least 3 days, it can be standardized and can be used in various experimental designs. Moreover, with different antibodies, it can detect different RNA modifications27.
It is important to separate different RNAs when analyzing RNA modifications. Ribosomal RNA comprises a large portion of the total amount of RNA28,29. The results from analyzing RNA modifications only in total RNA will represent mostly the changes of rRNA. Methylation and other such modifications of rRNA could potentially mask the changes in other RNAs. With the procedure of gel separation, the modifications of mRNA and other small RNAs can be more accurately analyzed.
Transcriptome-wide mapping with m6A immunoprecipitation and sequencing provides detailed insight into the modification of each type of RNA22. It provides information on the specific RNAs and a resolution of around 80-120 bp. Although m6A-Seq can compare the modifications between different experimental conditions, the selection of proper standards and controls for such experiments is difficult18. Immunoprecipitation is difficult to reproduce, often giving significant variations amongst repeats. Moreover, m6A-Seq requires the fragmentation of RNA samples before immunoprecipitation and sequencing30. The fragmentation process could potentially induce undue influences on the original RNA modifications. If the experiment does not need the specific gene's information but requires different conditions for comparison, the current method provides better visualization and control for diverse experimental setups. 
Modification of RNA is an important step in regulating transcriptional control31. However, the homeostasis and the regulatory mechanisms of various RNA modifications under diverse physiological realms are still unclear. Using the present modified northern blotting method, different RNA modifications can be quantified and compared. Furthermore, the changes and regulations of RNA modifications can be investigated in greater detail. In the future, it could also be possible to combine the experimental data from both the classical northern blotting and the modified northern blotting protocols, providing greater insights into RNA biology.
The most important factor determining the success of the modified northern blotting protocol is the integrity of the RNA sample. RNAs with some amount of degradation may yield good classical northern blotting results, but this could potentially have significant impact on the modified northern blotting results. The modifications of RNA in different tissues or cell lines could also vary significantly. It is important to test the suitable RNA sample loads for different tissues before performing the final experiments.
As blotting procedures have been traditionally named after Dr. Southern and different geographical directions, we propose the name "northeastern" blotting for the current technique.

Dynamic and reversible RNA modifications have important roles in RNA homeostasis. Four decades ago, N6-methyladenosine (m6A) modifications were found to be abundant in eukaryotic transcriptomes1. They have diverse functions in messenger RNA (mRNA), ribosomal RNA (rRNA), small nucleolar RNA, transfer RNA, and microRNA2. The m6A modifications of mRNA influence their splicing3, translation4, degradation5, and localization2. Moreover, they affect ribosome biogenesis and microRNA function6. The evolutionary conservation of m6A modifications of RNA is noted in unicellular bacteria to multi-cellular humans7. Delineation of the roles of m6A modifications is currently under extensive exploration. The efforts are expected to provide new insights into transcription control. Recent studies reveal that other chemical modifications of mRNA8 also play critical roles in RNA metabolism. 
Circadian rhythms9, stem cell pluripotency10, triglyceride metabolism4, fibrosis11, obesity12, and major depression13 are a few examples of processes where m6A modifications are known to control outcomes. Many circadian clock gene transcripts have m6A sites14. Modulation of m6A methylase or demethylase elicits circadian period changes15. Mettl3, an m6A transferase, is a regulator for stem cell pluripotency. A deficiency of Mettl3 leads to early embryonic lethality and aberrant lineage priming at the post-implantation stage10. A deficiency of fat mass- and obesity-associated (FTO), an m6A demethylase, in adipocytes affects fatty acid mobilization and body weight through posttranscriptional regulation of Angptl44. These studies reveal that m6A not only controls mRNA processing, but also plays critical roles in embryological development and patho-physiology. The function of m6A modifications holds implications for therapeutic considerations in the future.
Several methods are available to measure m6A modifications of RNA16-18. Traditionally, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are used to study the distribution of m6A in several RNAs19,20. Mass spectrometry is a sensitive tool for the detection of m6A modifications in RNA. However, the RNA needs to be excised by RNase into short fragments before analysis by mass spectrometry21. Methyl-RNA immunoprecipitation and sequencing (m6A-Seq)22 immunoprecipitates fragmented RNAs with m6A-specific antibodies and performs parallel RNA sequencing. This method generates transcriptome-wide m6A landscapes. High-resolution mapping of m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) further maps m6A modifications at a single-nucleotide resolution23. Both methods provide details of m6A modification across the whole transcriptome, with specific genes' information. However, quantifications and standardization in both methods are difficult if experiments require the comparison of multiple conditions. Moreover, fragmentations of RNA for m6A-Seq alter the original RNA structure, which may affect native m6A levels. To detect global m6A modifications of RNA and their changes under different experimental conditions, we report a method that employs a modified northern blotting protocol. This method resolves RNA by molecular weight, using gel electrophoresis18. This procedure provides better standardization and quantifications for experiments that involve multiple conditions or samples. It also provides specific m6A modification information for different RNAs, whether rRNA, mRNA, or microRNA.

<table><tbody><tr><td>RNaseZap solution</td><td>Ambion</td><td>AM9782</td><td>Protocol 1.1</td></tr><tr><td>TRI Reagent solution</td><td>Ambion</td><td>AM9738</td><td>Protocol 1.2.1.1</td></tr><tr><td>1-Bromo-3-chloropropane (BCP)</td><td>Sigma</td><td>B9673</td><td>Protocol 1.2.1.3</td></tr><tr><td>Ethanol</td><td>JTbaker</td><td>8006</td><td>Protocol 1.2.1.3</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>I9516</td><td>Protocol 1.2.1.3</td></tr><tr><td>GenElute mRNA Miniprep Kit</td><td>Sigma</td><td>MRN70</td><td>Protocol 1.2.2.1</td></tr><tr><td>Glycogen</td><td>Ambion</td><td>AM9510</td><td>Protocol 1.2.2.2</td></tr><tr><td>Sodium acetate</td><td>Fluka</td><td>71183</td><td>Protocol 1.2.2.2</td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9930</td><td>Protocol 1.2.2.6</td></tr><tr><td>DEPC</td><td>Sigma</td><td>D5758</td><td>Protocol 2.1.1</td></tr><tr><td>Agarose</td><td>JT Baker</td><td>A426</td><td>Protocol 2.1.2</td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td>Protocol 2.1.3</td></tr><tr><td>37% formaldehyde Solution</td><td>Sigma</td><td>F8775</td><td>Protocol 2.1.3</td></tr><tr><td>EDTA, Disodium Salt</td><td>JT Baker</td><td>8993</td><td>Protocol 2.1.3 10x MOPS buffer</td></tr><tr><td>formamide</td><td>Sigma</td><td>F7503</td><td>Protocol 2.2.1</td></tr><tr><td>RNA Millennium Marker</td><td>Ambion</td><td>AM7150</td><td>Protocol 2.2.2</td></tr><tr><td>Ethidium Bromide</td><td>Amresco</td><td>X328</td><td>Protocol 2.2.5</td></tr><tr><td>Ficoll PM400</td><td>GE Healthcare</td><td>17-0300-10</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>114391</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Xylene Cyanol FF</td><td>Sigma</td><td>X4126</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Sodium chloride dihydrate</td><td>JT Baker</td><td>3624</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Trisodium citrate</td><td>Sigma</td><td>S1804</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Hybond Blotting Paper</td><td>GE Healthcare</td><td>RPN6101M</td><td>Protocol 2.4.4</td></tr><tr><td>GE Hybond-N+ membrane</td><td>GE Healthcare</td><td>RPN303B</td><td>Protocol 2.4.6</td></tr><tr><td>Stratalinker UV Crosslinker 2400</td><td>Stratagene</td><td>400075</td><td>Protocol 3.1.2</td></tr><tr><td>Gel Catcher 1500</td><td>ANT Technology</td><td>Gel Catcher 1500</td><td>Protocol 3.1.5</td></tr><tr><td>Anti-m&lt;sup&gt;6&lt;/sup&gt;A (N&lt;sup&gt;6&lt;/sup&gt;-methyladenosine)</td><td>Synaptic Systems</td><td>202003</td><td>Protocol 3.2.3</td></tr><tr><td>Amersham ECL Anti-Rabbit IgG, HRP-linkd whole Ab</td><td>GE Healthcare</td><td>NA934</td><td>Protocol 3.2.5</td></tr><tr><td>ChemiDoc MP System</td><td>BIO-RAD</td><td>1708280</td><td>Protocol 3.2.8</td></tr></tbody></table>

a method for measuring rna n6-methyladenosine modifications in cells and tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

Normal ışık-karanlık sirkadiyen fazda 14 gün sonra, vahşi tip farelerde sürekli karanlıkta yerleştirildi. karaciğer RNA&#39;lar 4 saat aralıklarla numune ve modifiye Northern blot ile incelenmiştir. RRNA, mRNA ve küçük RNA&#39;ların metilasyonu açık (Şekil 2) tespit edilmiştir. Farklı sirkadiyen saatler arasında karşılaştırma (BT) doğru 18S rRNA standardı ile hesaplanabilir. RRNA, mRNA ve küçük RNA&#39;lar m 6 A düzeylerinin sağlam sirkadiyen salınım oldu. rRNA tarafından Sunulan girişimi önlemek için, poliadenile edilmiş RNA&#39;lar aşama 1.2.2 gibi saflaştırılabilir. Saflaştırmadan sonra, rRNA, büyük ölçüde diğer RNA&#39;lar (Şekil 3) daha iyi bir görünüm için izin ortadan kaldırılabilir. <img alt="Şekil 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt; Şekil 1: leke aktarma ünitesi montajı. zara RNA kurutma için kılcal aktarım ünitesi gösterilmektedir. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> Şekil 2: C57BL / 6J fareler m 6 A düzeyleri günlük ritim. Bu rakam, m 6 vahşi tip farelerin karaciğer toplam RNA&#39;nın bir leke 4 saat aralıklarla normal ışık-karanlık fazın 14 gün sonra koyu, koyu fazının ilk gününde kurban gösterir. Miktar m 6 18S ve 28S rRNA arasındaki görüntü yoğunluk farkı kullanılarak yapıldı. M 6 A bolluk altındaki formaldehit jelden 18S rRNA bandına normalize edildi.et = &quot;_ blank&quot;&gt; bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız. <img alt="Şekil 3," src="/files/ftp_upload/54672/54672fig3.jpg" /> Şekil 3: ya rRNA olmadan m 6 A lekeler. Örnek m toplam RNA ve vahşi tip fare karaciğerinde poliadenile RNA 6 bir blotlar gösterilmiştir. <a href="http://ecsource.jove.com/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1 </td><td colspan="3"> 10x MOPS tampon (pH 7.0, ışıktan koruma) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> MOPS </td><td> 41.85 </td><td> g </td><td> 0.2 M </td></tr><tr><td></td><td colspan="3"> Sodyum asetat </td><td> 4.1 </td><td> g </td><td>0.05 M </td></tr><tr><td></td><td colspan="3"> EDTA, disodyum tuzu </td><td> 3.7 </td><td> g </td><td> 0.01 M </td></tr><tr><td></td><td colspan="3"> DEPC su </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Genel Toplam </td><td> 1 </td><td> L </td><td></td></tr><tr><td></td><td colspan="3"> oda sıcaklığında karışmaya </td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> 20x SSC tamponu (pH 7.0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Sodyum klorit </td><td> 175.3 </td><td> g </td><td> 3 M </td></tr><tr><td></td><td colspan="3"> trisodyum sitrat </td><td> 88.3 </td><td> g </td><td> 0.3 M </td></tr><tr><td></td><td colspan="3"> DEPC su </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Genel Toplam </td><td> 1 </td><td> L </td><td></td&gt; </tr><tr><td></td><td colspan="3"> Oda sıcaklığında karıştırdıktan sonra otoklav </td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3"> takip Boya </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x MOPS tamponu </td><td> 500 </td><td> ul </td><td> 1x </td></tr><tr><td></td><td colspan="3"> Ficoll 400 </td><td> 0.75 </td><td> g </td><td> % 15 </td></tr><tr><td></td><td colspan="3"> mavi Bromophenol </td><td> 0.01 </td><td> g </td><td> % 0.2 </td></tr><tr><td></td><td colspan="3"> Ksilen siyanol </td><td> 0.01 </td><td> g </td><td> % 0.2 </td></tr><tr><td></td><td colspan="3"> DEPC su </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Genel Toplam </td><td> 5 </td><td> mL </td><td></td></tr><tr><td></td><td colspan="3"> -20 ° C&#39;de de </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> DEPC su </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 1&#39;den oranı sulandırmak: DEPC ve 1,000 GKD 2 O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> Gece boyunca oda sıcaklığında karıştırdıktan sonra otoklav </td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3"> Örnek tamponu (ışıktan korumak) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10x MOPS tamponu </td><td> 200 </td><td> ul </td><td></td></tr><tr><td></td><td colspan="3"> % 37 formaldehid </td><td> 270 </td><td> ul </td><td></td></tr><tr><td></td><td colspan="3"> formamid </td><td> 660 </td><td> ul </td><td></td></tr><tr><td></td><td colspan="3"> -20 ° C&#39;de de </td><td></td><td></td><td></td></tr></tbody&gt; </table> Tablo 1: Tampon ve çözümleri.

Watch this Scientific Journal Video about A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues at JoVE.com

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

RNA N 6 -methyladenosine (m 6 A) değişiklikleri ölçmek için bir değiştirilmiş Northern lekeleme yöntemi tarif edilmektedir. Geçerli yöntem çeşitli deneysel tasarımlar altında çeşitli RNA&#39;lar modifikasyonlar ve kontrolleri tespit edebilir.

RNA ölçülmesi için bir yöntem<em&gt; K</em<sup&gt; 6</supHücre ve dokularda&gt; -methyladenosine Değişiklikler

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

a-method-for-measuring-rna-n6-methyladenosine-modifications-cells

Research

JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

10.8K Görüntüleme.  Chang Gung Memorial Hospital, Chang Gung University College of Medicine.  RNA N 6 -methyladenosine (m 6 A) değişiklikleri ölçmek için bir değiştirilmiş Northern lekeleme yöntemi tarif edilmektedir. Geçerli yöntem çeşitli deneysel tasarımlar altında çeşitli RNA'lar modifikasyonlar ve kontrolleri tespit edebilir. 

Video: A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

TRNA-Isopentenyl transferaz aktivite tespit etmek için bir In Vitro tahlil

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Biyokimya

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

RNA metiltransferaz aktivite analizi için Antibody Içermeyen tahlil

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Genetik

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

Küçük RNA-seq iyileştirilmesi: Daha Az Önyargı ve 2'-O-Metil RNA'ların Daha İyi Tespiti

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

Arabidopsis'te m5C Modifikasyonu Çalışmak için Metillenmiş RNA İmmün Prempripitasyon Tahlil

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

<meta charset="utf8"></head><body>2D-HELS MS Seq: Farklı Nükleotid Modifikasyonlarına Sahip RNA Karışımlarının Doğrudan ve de novo Dizilimi için Genel LC-MS Tabanlı Bir Yöntem

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

2D-HELS MS Seq: Farklı Nükleotid Modifikasyonlarına Sahip RNA Karışımlarının Doğrudan ve de novo Dizilimi için Genel LC-MS Tabanlı Bir Yöntem

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Arginin Metilomunun Nükleer Manyetik Rezonans Spektroskopisi ile Araştırılması

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Kanser Araştırmaları

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

HPLC-MS/MS ile Nükleozid/Nükleotid İçerik Ölçümü için Bitki Numunesi Hazırlama

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

RRNA 2'-O-Metilasyonun DNAzyme bağımlı analizi

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

Gelişim Biyolojisi

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

Kütle Spektrometrisi Kullanarak Tek Nöronlardaki RNA Modifikasyonlarını Karakterize Etme

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

RNA ölçülmesi için bir yöntem

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