Biz bu protokolün geliştirilmesi sırasında hücre sıralama yapmadan onu yardım için CCR ETIB Sitometrisi Core V. Kapoor teşekkür etmek istiyorum. Biz de bu protokolün geliştirilmesi sırasında qPCR performans onların yardım M. Raffeld ve CCR LP Moleküler Tanı Ünitesi ve J. Zhu ve NHLBI DNA Dizilimi ve Genomik Çekirdek teşekkür ederiz. Bu araştırma NIH İntramural Programı tarafından desteklenmiştir.

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The authors have nothing to disclose.

Bu kültür içinde yetiştirilen yapışan hücreler popülasyonundan tek tek memeli hücrelerini izole etmek için, her hücrede yaklaşık 96 gen ekspresyonunu analiz etmek için bir yöntem sunulmuştur. Bu yöntem iyi çalışması için iyi bir ön hazırlık çok önemlidir. primerler tek hücreli ölçümlerin kalitesini belirlemek olarak, özellikle ilgi transkriptleri (1.2-1.3 adımlar) özgü tasarımı ve test primer çiftleri, zaman alıcı ama önemli adımlardır. güvenilir bir primer çiftleri elde edil...

Nüfusu tek tek hücrelerin tek tip bir fizyolojik uyarıcı, 1, 2, 3, 4 çok farklı tepkiler gösterebilirler. Bir popülasyonda hücrelerin genetik varyasyonu bu tepkilerin çeşitli mekanizmalardan biri olmakla birlikte, daha hücre klonal popülasyonunda, yanıtların çeşitliliğini artırmak olmayan çeşitli genetik faktörler de vardır. Örneğin, tek tek proteinlerin ve diğer önemli sinyal molekülleri seviyeleri, aşağı doğru gen sentezleme profillerinin değişimine yol açan, bir hücre tarafından hücre bazında değişebilir. Buna ek olarak, gen aktivasyon kaymasının 7, 8, 9 her transkript, nispeten az sayıda sınırlı olabilir transkript 5, 6 kısa süreli patlamaları oluşabilir. böyleGen aktivasyon stochasticity büyük ölçüde biyolojik yanıt değişikliklere katkıda ve fizyolojik bir uyarıcıya yanıt mikroorganizmalar 10 ve memeli hücrelerinde 1 2 seçici bir avantaj sağlayabilir. Nedeniyle değişkenlik hem genetik hem de genetik olmayan kaynaklara, bir uyarıcıya tepki olarak belirli bir hücre gen tanımlama profili dökme yanıtın ölçülmesi elde edilen ortalama gen tanımlama profili büyük ölçüde farklılık gösterebilir. tek tek hücreler bir uyarıcıya tepki olarak değişkenlik göstermektedir ne ölçüde belirlenmesi tek tek hücrelerin izole edilmesi için teknikler gerektirir, ilgi konusu transkript için ekspresyon seviyelerinin ölçülmesi, ve elde edilen sentezleme verilerinin sayısal analizi. maliyetleri geniş bir kaplama, tek bir hücre içinde gen ekspresyonunu analiz etmek için çeşitli yaklaşımlar bulunmaktadır transkriptlerin sayısı problanmış veölçümü doğruluğu. Örneğin, tek hücreli RNA Seq transkript içerisinde ve tek tek hücreler en yüksek ölçüde sentezlenen genler için farklı transkript binlerce ölçmek için yeteneği geniş bir derinliğe sahiptir; maliyetler düşmeye devam rağmen, ancak böyle bir sıralama derinliği ile ilişkili maliyet, engelleyici olabilir. Bunun aksine, in situ hibridizasyon (smRNA FISH), tek bir molekül, RNA floresan için transkriptlerin kesin miktarının sunar daha düşük ifade ilgili gen başına makul bir maliyetle genleri; Bununla birlikte, hedef genlerin sadece az sayıda bu yaklaşım, belirli bir hücre içinde analiz edilebilir. Bu protokol açıklanan kantitatif PCR bazlı deneyler, bu teknikler arasında bir orta yol sağlar. Bu deneyler kadar 96 hücrelerinde aynı anda ilgi 96 transkript kadar ölçmek için mikroakışkan gerçek-zamanlı PCR makinesi kullanmaktadır. Yukarıda bahsedilen yöntemlerin her biri, gerekli donanım maliyetlerini olsa da, herhangi bir tek tek qPCR tahlilinde maliyeti nispetendüşük. Bu protokol, mikroakışkan gerçek-zamanlı PCR makinesi üreticisi (Protokol ADP 41, Fluidigm) önerdiği bir uyarlanmıştır. PCR-temelli yaklaşımla her transkript mutlak sayısının tahmini etkinleştirmek için, birden fazla deneyler arasında kullanılabilir Hazırlanan hedef gen amplikonlarının iç kontrollerin faydalanmak için protokol genişlettik. Bu tekniğin bir örneği olarak, MCF-7 insan göğüs karsinoma hücrelerinde tümör baskılayıcı p53&#39;ün düzenlenen genlerin ifadesinin ölçümü 11 tarif edilmiştir. Hücreler DNA çift iplikli tatili uyaran bir kimyasal madde ile zorlanmaktadır. Daha önceki çalışmalar, DNA çift iplikli kırılmalara p53 yanıt hem de p53 seviyeleri 12 açısından ve farklı hedef genlerin 11 aktivasyonu tek tek hücrelerin heterojenlik büyük bir sergilediğini göstermiştir. Ayrıca, p53 100 ekspresyonunu düzenleyenHücre döngüsü tutuklama, apoptozi ve yaşlanma 13, 14 dahil olmak üzere birçok aşağı akış yolakları, yer alan hedef genlerin iyi karakterize edilir. Her bir hücrede p53 aracılı tepki kompleksi hem değişken hem de olduğu bir yaklaşım sistem faydaları analizi teknikleri, yaklaşık 100 hedef genler, aşağıda anlatıldığı gibi, tek tek hücrelerin, eş zamanlı olarak incelenebilir. (Örneğin, tek hücreli izolasyonu ve liziz alternatif yöntemler olarak) hafif değişiklikler, protokol kolaylıkla memeli hücre tipleri, transkriptlerin ve hücresel tepkilerin bir geniş bir çalışma için adapte edilebilir. Uygun önceden hazırlık, hücre sıralama ve gen ifadesi ölçüm yuvarlak üç günlük bir süre boyunca bu protokole göre yapılabilir. Aşağıdaki zamanlama önerilmektedir: önceden, ilgi transkript seçin belirlemek ve bu TRANSCR cDNA yükseltmek primer çiftleri doğrulamakIPTS ve standartları ve bu primerler kullanılarak primer karışımları hazırlayın. Gün 1, hücre tedavisi, hasat takip ve hücreleri sıralamak, ters transkripsiyon ve belirli hedef amplifikasyon gerçekleştirmek ve adi primerler kaldırmak için bir eksonükleaz ile örnekleri davranın. 2. Gün, qPCR kullanılarak sıralanır hücreleri üzerinde kalite kontrolünü yapmak. Son olarak, 3. gün, mikroakışkan qPCR kullanılarak sıralanır hücrelerinde gen ifadesini ölçün. Şekil 1 ilgili adımları özetlemektedir.

<table border="0" cellpadding="0" cellspacing="0" width="988">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:488px;">RNeasy Plus Mini Kit</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:147px;">74134</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>ThermoFisher</td>
			<td>4374966</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phusion High-Fidelity DNA Polymerase</td>
			<td>New England BioLabs</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quant-iT High-Sensitivity dsDNA Assay Kit</td>
			<td>ThermoFisher</td>
			<td>Q33120&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.0-mL low adhesion microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1420-2600</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA Suspension Buffer</td>
			<td>Teknova</td>
			<td>T0221</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Axygen 0.2-mL Maxymum Recovery Thin Wall PCR Tubes</td>
			<td>Corning</td>
			<td>PCR-02-L-C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GE 96.96 Dynamic Array DNA Binding Dye Sample &#38; Assay Loading Reagent Kit</td>
			<td>Fluidigm</td>
			<td>100-3415</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone RPMI 1640 media</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum, Certified (US)</td>
			<td>ThermoFisher</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic-Antimycotic Solution</td>
			<td>Corning</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neocarzinostatin</td>
			<td>Sigma</td>
			<td>N9162</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ELIMINase</td>
			<td>Decon Labs</td>
			<td>1101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SUPERase-In</td>
			<td>ThermoFisher</td>
			<td>AM2696</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CellsDirect One-Step qRT-PCR Kit</td>
			<td>ThermoFisher</td>
			<td>11753500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E. coli DNA</td>
			<td>Affymetrix</td>
			<td>14380 10 MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ThermalSeal Sealing Film, Sterile</td>
			<td>Excel Scientific</td>
			<td>STR-THER-PLT</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD FACSAria IIu</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Trypsin 0.05%</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30236.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, 1x</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon 40&#181;m Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Exonuclease I</td>
			<td>New England BioLabs</td>
			<td>M0293S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SsoFast EvaGreen Supermix with Low ROX</td>
			<td>Bio-Rad</td>
			<td>172-5210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96.96 Dynamic Array IFC for Gene Expression (microfluidic qPCR chip)</td>
			<td>Fluidigm</td>
			<td>BMK-M-96.96</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IFC Controller HX (loading machine)</td>
			<td>Fluidigm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BioMark or BioMark HD (microfluidic qPCR machine)</td>
			<td>Fluidigm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Real-Time PCR Analysis software&#160;</td>
			<td>Fluidigm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB software</td>
			<td>MathWorks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell gene expression profiling using facs and qpcr with internal standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

Protokol genel bir bakış hücre tedavisi, FACS ile tek hücrelerin izolasyonu için adımları da dahil olmak üzere Şekil 1 &#39;de gösterilen üretim ve tek hücre lizatları, tek hücreli bir cDNA kütüphanelerinin onay cDNA kütüphanelerinin ön amplifikasyon sıralı oyuklar ve qPCR gen ifadesinin ölçümü. tek hücre izolasyonu ve gen ekspresyon analizi için hazırlanırken, ilk önce her bir il...

Watch this Scientific Journal Video about Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards at JoVE.com

Dahili Standartlarla FACS ve qPCR Kullanarak Tek Hücreli Gen İfadesi Profili Oluşturma | Genetik | Video

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

İç Standartları FACS ve qPCR kullanma Tek hücreli Gen İfadesi Profil

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

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16.3K Görüntüleme.  National Cancer Institute. We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Video: Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

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

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

İrritabl Bağırsak Sendromu içinde miRNA&#39;lar Sirkülasyon tedirginlikler bir Çoklanmış Yüksek verim Gen İfadesi Platformu Kullanma Algılanan

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

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 ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Tek Hücre sıralama kullanarak Kopya sayısı Değişmeler Algılama

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

Kantitatif Gerçek Zamanlı PCR Kullanarak Sıçanların Periton Membranındaki MikroRNA İfadesinin Saptanması

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

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 ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Nesil bir gen kesintiye Streptococcus mutans çoğunlukla zorlanma olmadan Gen Klonlama

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

Gen ifadesinin artan işlem hacmi ile alı arpa Aleurone katmanlarında ölçme

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Metabolik etiketleme ve Transfer RNA Macroarrays kullanarak profil oluşturma

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...