Biz İşlevsel Genomik Tesisi ve spektrografisi ve Biyogörüntüleme Tesisi, NIBB Çekirdek Araştırma Hizmetleri ve Model Bitki Araştırma Tesisi, NIBB Bıoresource Merkezi&#39;nin çalışanlarına teşekkür ederiz.

Bitki tohumları toplam RNA&#39;nın 1. Ekstraksiyon <ol><li> tampon setleri, spin sütunları, 1.5 ve 2.0 ml polipropilen tüpler ve nükleaz içermeyen 1.5 mL polipropilen tüpleri hazırlayın. </li><li> (Ağırlık / hacim) (bundan sonra PVP olarak anılacaktır) moleküler biyoloji sınıf polivinilpirolidon kuvvetli RNA ekstraksiyonu ve vorteks için lizis tamponu hücre% 1 ekleyin. tamamen çözülmesi, 25 ° C&#39;de 20 dakika süreyle inkübe edin. 20 dakika inkübasyondan sonra, kabarcıkların oluşumunu önlemek için ters tüp çevirerek hafifçe tampon karıştırın. Kullanmadan önce oda sıcaklığında (15-25 ° C) muhafaza ediniz. </li><li> Buz üzerinde 1.5 ml polipropilen tüpleri içinde Arabidopsis thaliana bitkileri ve yerden hasat meyveleri. alüminyum plakalar üzerine yerleştirin meyve 4 ° C&#39;de muhafaza ve stereo mikroskop altında meyve tohumlarını izole. <ol><li> Sıvı n tüpleri hemen buz üzerine bir alüminyum rafın saklanan 1.5 ml polipropilen tüplere izole tohumları (yaklaşık olarak 200 tohum) yerleştirin veitrogen. </li></ol></li><li> sıvı azot tüpleri çıkarın ve buz üzerinde alüminyum raf onları geri. 4 ° C&#39;de 1000 x g&#39;de 1% 1 dakika için PVP ve santrifüj ihtiva eden tampon 100 ul ekle. </li><li> Buz üzerinde, alüminyum rafın tüp tutulurken 60 saniye için bir motor-öğütücü kullanılarak paslanmaz çelik bir havan tokmağı ile homojenize örnek. </li><li> % 1 PVP içeren tampon 550 mcL ters tüp çevirerek tampon hafifçe karıştırın ve 25 ° C&#39;de 10 dakika inkübe edilir. </li><li> 5 25 ° C&#39;de 8000 x g&#39;de dakika ve yeni bir 1.5 ml polipropilen tüpe Süpernatant 550 uL santrifüjleyin. </li><li> 5 25 ° C&#39;de 10,000 x g&#39;de dakika ve yeni bir 1.5 ml polipropilen tüpe Süpernatant 450 uL santrifüjleyin. </li><li> RNA ekstraksiyonu için hücre lizatı gibi süpernatant kullanın. Bundan sonra, piyasada mevcut olan kit üreticinin talimatlarında açıklanan prosedürü uygulayın. </li><li> ZehirRNA, düşük seviyede transkriptlerinin ekspresyonu kontrolü için RNA yeterince yüksek bir konsantrasyonunu sağlamak için elüsyon tamponunun minimum hacim kullanılarak gerçekleştirilmiştir. kullanılıncaya kadar derin dondurucuda RNA saklayın. </li></ol> RNA Kalite 2. Doğrulama <ol><li> Toplam RNA çözülme nazik dokunarak karıştırın ve buz üzerinde bir alüminyum rafa tüp yerleştirin. </li><li> Bir microvolume spektrofotometre kullanılarak RNA konsantrasyonu, A260 / A280 oranı ve A260 / A230 oranı ölçün. </li><li> 70 ng / ml bir son konsantrasyona kadar, RNaz içermeyen su içinde toplam RNA seyreltilir. </li></ol> Tohumlar toplam RNA&#39;nın 3. Ters Transkripsiyon <ol><li> ticari kit toplam RNA ve tampon setleri çözülme. nazik dokunarak sonra buz üzerinde kiti enzimleri tutun. nükleaz içermeyen 0.2 ml polipropilen tüpleri hazırlayın. </li><li> 0.2 mL Polipropilen 5 toplam RNA uL, 1 uL Oligo dT primeri (50 uM) ve 1 Random uM 6-mer (50 uM) ilavebuz üzerinde lene tüp. </li><li> 37 ° C &#39;de 15 dakika boyunca inkübe ve buz üzerine yerleştirin. </li><li> Bundan sonra, ters transkripsiyon-PCR kiti üreticinin talimatlarında açıklanan prosedürü uygulayın. </li><li> Kullanmadan önce buz üzerinde ters transkripsiyon ürünlerini yerleştirin. </li></ol> 4. Kantitatif Real-time PCR Analizi <ol><li> üreticinin protokolünü kullanarak, hedef gen dizilerini barındıran plasmidleri oluşturun. </li><li> standart eğrileri için DNA şablonları 100-500,000 kopya / uJ&#39;ye konsantrasyonları ayarlayın. </li><li> damıtılmış suyla: cDNA çözümleri (100 1) seyreltin. </li><li> 2 seyreltilmiş cDNA çözümleri uL ve kantitatif real-time PCR kiti master karışımı standart eğriler için plazmidler ekleyin. </li><li> 30 sn için 95 ° C ve daha sonra 5 saniye boyunca 95 ° C &#39;de 40 döngü 35 sn için 60 ° C: Aşağıdaki bisiklet koşulları kullanılarak gerçek zamanlı PCR ayarlayın. </li><li> adama göre kopya sayısını analizGerçek zamanlı PCR sistemi olarak üreticinin talimatları. </li></ol>

<ol>
	<li>Hills, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+storage-product+synthesis+in+seeds.">Control of storage-product synthesis in seeds.</a> Curr Opin Plant Biol. 7 (3), 302-308 (2004).</li><li>Li-Beisson, 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=Acyl-lipid+metabolism.">Acyl-lipid metabolism.</a> Arabidopsis Book. 11, e0161 (2013).</li><li>Bates, P. D., Stymne, S., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+pathways+in+seed+oil+synthesis.">Biochemical pathways in seed oil synthesis.</a> Curr Opin Plant Biol. 16 (3), 358-364 (2013).</li><li>Santos-Mendoza, 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=Deciphering+gene+regulatory+networks+that+control+seed+development+and+maturation+in+Arabidopsis.">Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis.</a> Plant J. 54 (4), 608-620 (2008).</li><li>Durrett, T. P., Benning, C., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+triacylglycerols+as+feedstocks+for+the+production+of+biofuels.">Plant triacylglycerols as feedstocks for the production of biofuels.</a> Plant J. 54 (4), 593-607 (2008).</li><li>Kanai, 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=The+Plastidic+DEAD-box+RNA+helicase+22,+HS3,+is+essential+for+plastid+functions+both+in+seed+development+and+in+seedling+growth.">The Plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.</a> Plant Cell Physiol. 54 (9), 1431-1440 (2013).</li><li>Kanai, 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=Extension+of+oil+biosynthesis+during+the+mid-phase+of+seed+development+enhances+oil+content+in+Arabidopsis+seeds.">Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds.</a> Plant Biotechnol J. 14 (5), 1241-1250 (2016).</li><li>Dekkers, B. J., 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=Identification+of+reference+genes+for+RT-qPCR+expression+analysis+in+Arabidopsis+and+tomato+seeds.">Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds.</a> Plant Cell Physiol. 53 (1), 28-37 (2012).</li><li>Salzman, R. A., 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=An+improved+RNA+isolation+method+for+plant+tissues+containing+high+levels+of+phenolic+compounds+or+carbohydrates.">An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates.</a> Plant Mol Biol Rep. 17 (1), 11-17 (1999).</li><li>Vicient, C. M., Delseny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+total+RNA+from+Arabidopsis+thaliana+seeds.">Isolation of total RNA from Arabidopsis thaliana seeds.</a> Anal Biochem. 268 (2), 412-413 (1999).</li><li>Wang, G. F., 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=Isolation+of+high+quality+RNA+from+cereal+seeds+containing+high+levels+of+starch.">Isolation of high quality RNA from cereal seeds containing high levels of starch.</a> Phytochem Analysis. 23 (2), 159-163 (2012).</li><li>Birtic, S., Kranner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-quality+RNA+from+polyphenol-,+polysaccharide-+and+lipid-rich+seeds.">Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds.</a> Phytochem Analysis. 17 (3), 144-148 (2006).</li></ol>

The authors have nothing to disclose.

Gen ekspresyon profilleri bitki fizyolojisinin anlayışımıza katkıda; Bu nedenle, belirli bir RNA izolasyon yöntemleri her bir örnek durum 9-12 geliştirilmiştir. Tohumları RNA izolasyonu sırasında inhibe ve RNA silika membranlanna bağlanan ölçüde inhibe olduğu bulundu işlemleri incelenmiştir. Yağ, proteinler ve polifenoller büyük miktarlarda RNA izolasyonu inhibe eder. Biz RNA silika membranların bağlanma işleminden önce bir parçalama solüsyonu ile bu bileşikleri kaldırmak için R...

Bitkiler nesil doğuran tohum üretmek. Tohumlar, post-germinatif büyüme yağlar, karbohidratlar ve proteinler gibi depolama rezervlerinin büyük miktarda birikir. İnsanlar gıda ve hayvan yemi kaynağı olarak tohum depolama rezervleri kullanmak ve böylece bitki tohumları dünya çapında yenilebilir organik maddenin önemli tedarikçilerinden biri vardır. tohum verimi artan bitki biliminde önemli bir sorundur. Tohum depolama rezervleri gıda ve endüstriyel malzemelerin ticari olarak değerli kaynaklar olduğundan, bu rezervlerin metabolizmasının düzenlenmesine altında yatan moleküler mekanizmalar yaygın 1-6 incelenmiştir. Bundan başka, bu mekanizmaların tanıtılması bitkileri tohum verimini artırmak için yararlı olacaktır. Tohumlar Döllenmeden sonra bitki yumurtalıklarda gelişir ve bunlar gelişim aşamaları 1,6,7 bir dizi olgun. Dahası, moleküler mekanizma yatan tohum gelişimini anlamak detaylı gerektirirTohum geliştirme bir dizi hassas gen ekspresyon profillerini üretilecek. Ancak, bitki tohumlarında yağlar, proteinler, karbohidratlar, ve polifenoller yüksek miktarlarda zor sentezlenmesinin hassas profil önleyen yüksek ölçüde saflaştırılmış RNA izole etmek istiyorum. Burada, sıvı yağlar, proteinler ve polifenoller büyük miktarlarda ihtiva eden yağlı tohumlardan RNA izolasyonu için etkili bir yöntem getirmektedir. Bu yöntemi kullanarak, araştırmacılar yüksek derecede arıtılmış RNA&#39;nın hazırlanması mümkün olacaktır. RNA geliştirilmesi ve olgun yağlı tohumlar tohum depo edilen metabolik kontrol düzenlenmesi önemli genlerin transkripsiyonel değişikliklerin izlenmesi için yararlı olacaktır.

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			<td height="21" style="height:21px;width:247px;">RNeasy Plant Mini Kit</td>
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			<td style="width:136px;">74904</td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">polyvinylpyrrolidone</td>
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			<td style="width:136px;">P5288-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HOMOGENIZER S-303</td>
			<td style="width:81px;">AS ONE</td>
			<td style="width:136px;">1-1133-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">NanoDrop Lite</td>
			<td style="width:81px;">Thermo Scientific</td>
			<td style="width:136px;">ND-NDL-US-CAN</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PrimeScript RT reagent Kit (Perfect Real Time)</td>
			<td style="width:81px;">TAKARA</td>
			<td style="width:136px;">RR037A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">KAPA SYBR Fast qPCR kit</td>
			<td style="width:81px;">Kapa biosystems</td>
			<td style="width:136px;">KK4601</td>
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</table>

an efficient method for the isolation of highly purified rna from seeds for use in quantitative transcriptome analysis

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

Biz ilk Arabidopsis olgun tohum kullanılarak PVP optimal konsantrasyonunu araştırdık. Toplam RNA,% 0,% 0.25,% 0.5,% 1.0 veya% 2.0 PVP içeren hücre parçalama tamponu kullanılarak, yukarıda tarif edilen protokole göre yaklaşık 1.000 tohumlardan izole edildi. Yağ tabakası ve tohum artıkları (Şekil 1A) kaçınarak homojenizasyon ve santrifüj sonrasında üst faz toplanmıştır. <img alt=...

Watch this Scientific Journal Video about An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis at JoVE.com

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

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

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

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

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Research

JoVE Journal

Biochemistry

9.6K Görüntüleme.  National Institute for Basic Biology. We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

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

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

Küçük RNA miktarının belirlenmesi Sigara kodlama için yüksek yoğunluklu lipoproteinler izolasyonu

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Biyokimya

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

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

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

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

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

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Kantitatif Kütle Spektrometresi Analiz lipid Damlacık izolasyonu

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Proteinler ve onların izoformlarının kapiller Isoelectric odaklanarak yöntemi tarafından son derece hassas ve nicel tespiti

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Demir içeriği tesislerinde ölçme kolorimetrik yöntemi

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

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

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

Çeviri başlatma karmaşık oluşumu memeli mRNA'ların Toeprinting analizi

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

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

Hızlı izole edilmesi HEK hücrelerden Mitoribosome

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Endotel hücrelerinden RNA yalıtım için Ribosome affinity arıtma (TRAP) çevirisi In vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

Gen Ekspresyonu Profilleme için Modifiye Yöntemi ile Tahıl Tohumlarından Yüksek Kaliteli Transkripsiyon Verilerinin Elde Edilmesi

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Kantitatif Transkriptom analizinde kullanım için tohumlar yüksek oranda saf RNA'nın izolasyonu için etkili bir yöntem

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