著者の謝辞があります。

<ol>
	<li>Aronson, S. J., Rehm, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+foundation+for+genomics+in+precision+medicine.">Building the foundation for genomics in precision medicine.</a> Nature. 526 (7573), 336-342 (2015).</li><li>Ball, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+GWAS:+power,+sample+size,+and+data+structure.">Designing a GWAS: power, sample size, and data structure.</a> Genome-Wide Association Studies and Genomic Prediction. , 37-98 (2013).</li><li>Kwon, J. M., Goate, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+candidate+gene+approach.">The candidate gene approach.</a> Alcohol research and health. 24 (3), 164-168 (2000).</li><li>Oeth, P., Mistro, G. D., Marnellos, G., Shi, T., van den Boom, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+genotyping+using+single+base+primer+extension+coupled+with+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry+(MassARRAY).">Qualitative and quantitative genotyping using single base primer extension coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MassARRAY).</a> Single Nucleotide Polymorphisms: Methods and Protocols. , 307-343 (2009).</li><li>Pusch, W., Kostrzewa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+MALDI-TOF+mass+spectrometry+in+screening+and+diagnostic+research.">Application of MALDI-TOF mass spectrometry in screening and diagnostic research.</a> Current pharmaceutical design. 11 (20), 2577-2591 (2005).</li><li>Su, K. 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=Pretreatment+epidermal+growth+factor+receptor+(EGFR)+T790M+mutation+predicts+shorter+EGFR+tyrosine+kinase+inhibitor+response+duration+in+patients+with+non-small-cell+lung+cancer.">Pretreatment epidermal growth factor receptor (EGFR) T790M mutation predicts shorter EGFR tyrosine kinase inhibitor response duration in patients with non-small-cell lung cancer.</a> Journal of clinical oncology. 30 (4), 433-440 (2012).</li><li>Singhal, N., Kumar, M., Kanaujia, P. K., Virdi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-TOF+mass+spectrometry:+an+emerging+technology+for+microbial+identification+and+diagnosis.">MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis.</a> Frontiers in microbiology. 6, 791 (2015).</li><li>Cricca, 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=High-throughput+genotyping+of+high-risk+Human+Papillomavirus+by+MALDI-TOF+Mass+Spectrometry-based+method.">High-throughput genotyping of high-risk Human Papillomavirus by MALDI-TOF Mass Spectrometry-based method.</a> New Microbiologica. 38 (2), 211-223 (2015).</li><li>Ashley, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Variation+at+the+Th2+Immune+Gene+IL13+is+Associated+with+IgE-mediated+Paediatric+Food+Allergy.">Genetic Variation at the Th2 Immune Gene IL13 is Associated with IgE-mediated Paediatric Food Allergy.</a> Clinical &amp; Experimental Allergy. 47 (8), 1032-1037 (2017).</li><li>Bousman, C. 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=Effects+of+NRG1+and+DAOA+genetic+variation+on+transition+to+psychosis+in+individuals+at+ultra-high+risk+for+psychosis.">Effects of NRG1 and DAOA genetic variation on transition to psychosis in individuals at ultra-high risk for psychosis.</a> Translational psychiatry. 3 (4), e251 (2013).</li><li>Moffatt, M. 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=A+large-scale,+consortium-based+genomewide+association+study+of+asthma.">A large-scale, consortium-based genomewide association study of asthma.</a> New England Journal of Medicine. 363 (13), 1211-1221 (2010).</li><li>Granada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+association+study+of+plasma+total+IgE+concentrations+in+the+Framingham+Heart+Study.">A genome-wide association study of plasma total IgE concentrations in the Framingham Heart Study.</a> Journal of Allergy and Clinical Immunology. 129 (3), 840-845 (2012).</li><li>Oeth, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPLEX+assay:+Increased+plexing+efficiency+and+flexibility+for+MassARRAY+system+through+single+base+primer+extension+with+mass-modified+terminators.">iPLEX assay: Increased plexing efficiency and flexibility for MassARRAY system through single base primer extension with mass-modified terminators.</a> Sequenom application note. 27, (2005).</li><li>Ellis, J. A., Ong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MassARRAY+System+for+Targeted+SNP+Genotyping.">The MassARRAY System for Targeted SNP Genotyping.</a> Genotyping: Methods and Protocols. , 77-94 (2017).</li><li>Johnson, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAP:+A+web-based+tool+for+identification+and+annotation+of+proxy+SNPs+using+HapMap.">SNAP: A web-based tool for identification and annotation of proxy SNPs using HapMap.</a> Bioinformatics. 24 (24), 2938-2939 (2008).</li></ol>

著者が明らかに何もありません。

ここでは、質量分析法による多重遺伝子型解析法を示します。代表的な結果は、MALDI-TOF 質量分析法4分析化学と材料表13に掲げると共に PCR を使用して生成されました。このプラットフォームでは、合計 40 時間以内 9 SNPs の 1,255 個人 11,295 遺伝子型をラボで生成されます。
生成し、食物アレルギーと独立した複製 phenotyped 探...

人間の複雑な疾患の遺伝的変異が疾患感受性に貢献、これらの亜種の定量化は理解病因、治療レスポンダーの高リスク患者のグループを識別するために有用かもしれない。確かに、精密医療の約束は患者サブグループ1を識別するためにゲノム情報を利用したに依存しています。残念ながら、どこ疾患表現型の相当な遺伝的多様性、低浸透度と変数表現力によって支えられている、複雑な病気の生物学領域内コホート サイズ ゲノム全体小説的なアプローチのための要件候補者は、しばしば膨大2です。また、標的候補遺伝子アプローチから始まる疾患病因3に固有の遺伝子/経路について事前の仮説。経路解析ツールは、探検するために多数の候補経路を生成する標的遺伝子座の病態生理学を調査するため使用されます。ここで人間のコホート研究4に適した 1 つの試金と Snp の数百人に数十の調査を可能にする多重遺伝子型アプローチを紹介します。このアプローチは比較的高スループット、数百、数千の新規探索研究誘因する DNA サンプルや特定経路の調査を許可します。ここで説明する方法は、比較的迅速かつ安価な方法で識別リスク対立遺伝子および臨床特徴との関連付けに役立ちます。このプラットフォームは、微生物感染症7ひと乳頭腫ウイルス8スクリーニングおよび診断目的5,6と最近では非常に有利にされています。
調査の結果、すなわち遺伝子のセットを選択から始まるこのプロトコル、文献検索、または病気プロセスに関与の先験的な仮説によって通常決定対象地域。または、おそらく選択した探索ゲノム広い連合 (GWA) 研究の主要な団体としてレプリケーション。遺伝子セットから研究者はタグ SNPs の洗練されたリストを選択します。つまり、連鎖不平衡 (LD)、または地域での亜種間で相関が高い LD、ハプロタイプと呼ばれる中の Snp のグループの代表的な 'タグを SNP を識別するために使用されます。地域の高い LD 継承を意味します、SNPs がしばしば一緒に遺伝子型 1 つの SNP がハプロタイプですべての Snp で変化を表すために十分になるよう。また、フォロー アップ SNPs の決定的なリストから多くの地域、例えば場合。、GWA 研究のレプリケーションでは、このプロセス必要がありますされません。多重遺伝子型は増幅プライマー拡張プライマーの質量が異なるは、解釈を生成する製品の質量スペクトル、アッセイは、これらのターゲットの周り設計されなければなりません。これらのパラメーターは、多重遺伝子型分析設計ツールによって簡単に実装されます。このデザインから前方および逆のプライマーは、SNP を含むシーケンスを増幅して興味のマーカーをターゲットに使用されます。拡張プライマーを直接 SNPs に近位付 SNP に補足である単一、大量更新、'ターミネーター' ベースが追加されます。ターミネーター ベース DNA の拡張をできなくなります。ベースの質量の変更により、質量分析による検出を 1 つのベースが異なるフラグメント。ジェノタイピング化学を含むプレート質量分析プラットフォームに測定用チップに適用されます。適切な品質管理をシステムによって検出された生ジェノタイピング呼び出しに適用すると、データをエクスポートし、疾患表現型との関連付けをテストするのには統計分析に使用できます。

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genomic DNA&#160;</td>
			<td style="width:127px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:235px;">1 &#956;L at a concentration of 5-10 ng/&#956;L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Primers: forward and reverse amplification and extension</td>
			<td style="width:127px;">IDT</td>
			<td style="width:119px;">-</td>
			<td style="width:235px;">see manuscript section 1.2.1 on design of primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionized water&#160;</td>
			<td style="width:127px;">E.g. Milli-Q water&#160;</td>
			<td style="width:119px;">-</td>
			<td>deionized with 18.2 M&#937;.cm resistivity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Genotyping reagent kit. iPLEX Gold Chemistry reagent set&#160;</td>
			<td>Agena Bioscience</td>
			<td style="width:119px;">#10148-2</td>
			<td style="width:235px;">includes all reagents for reactions in 2.2.1, 2.3.1 and 2.4.2 , chip and resin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR plates (384-well)</td>
			<td style="width:127px;">Abgene</td>
			<td style="width:119px;">#ABGAB-1384</td>
			<td style="width:235px;">For the MassARRAY system plates by Abgene are compatible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipettes</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>single and 8-channel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>compatible with 384-well plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Thermocycler</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:235px;">compatible with PCR programs as detailed in 2.2.4, 2.3.2 and 2.4.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimple resin plate&#160;</td>
			<td>Agena Bioscience</td>
			<td style="width:119px;"></td>
			<td>6mg, 384-well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate rotator&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MassARRAY Analyzer 4 System</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">MALDI-TOF (matrix-assisted laser desorption/ionization &#8211; time of flight) Mass Spectrometer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RS1000 Nanodispenser</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Assay Design Suite</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td>Tool used to design the multiplex genotyping assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot Start Taq</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>DNA polymerase enzyme&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resin&#160;</td>
			<td style="width:127px;">Agena Biosciences</td>
			<td style="width:119px;"></td>
			<td>Supplied with iPLEX kit</td>
		</tr>
	</tbody>
</table>

candidate gene testing in clinical cohort studies with multiplexed genotyping and mass spectrometry

複雑な病気の多くの場合疾患感受性に貢献する複数の一般的な遺伝子変異によって支えられています。ここでは、ジェノタイピング マルチプレックス アッセイ質量分析法を用いた臨床コホートで遺伝子経路の連合を調査するコスト効果の高いタグ単一ヌクレオチド多型 (SNP) アプローチについて述べる。例として食品アレルギー候補遺伝子座Interleukin13 (IL13) を調べた。このメソッドは、領域内の共有連鎖不平衡 (LD) を利用して効率的にカバレッジを最大化します。選択した LD SNPs、設計されています多重アッセイに同時に分析するまで 40 の異なる Snp の有効化費用対効果を高めます。ポリメラーゼの連鎖反応 (PCR) は単一のヌクレオチドの拡張子、ターゲット遺伝子を増幅するため使用され、amplicons マトリックス支援レーザー脱離/イオン化-時間 flight(MALDI-TOF) 質量分析装置を使用して測定されます。Raw 出力を厳格な品質管理の定義と切り取りトレードオフを使用してソフトウェアを呼び出す遺伝子で解析、確率が高い遺伝子型の決定しデータ分析のための出力します。

上記プロトコルを使用我々 は誤は食品アレルギーの場合とコントロール9のコホートにおける Th2 免疫遺伝子IL13の SNPs をタグ付け。関心領域内の遺伝的変異が食品アレルギー リスクを増加させるかどうかをテストするのには、祖先とその他の潜在的な共変量調整後のロジスティック回帰分析を行った。テーブル 109</su...

Watch this Scientific Journal Video about Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry at JoVE.com

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

多重遺伝子型及び質量分析法と臨床コホート研究でテスト候補遺伝子

複雑な人間の病気に貢献する遺伝的変異の同定機構を識別することができます。ここでは、候補者の遺伝子または遺伝子パスウェイ解析低コストでカバレッジを最大化、コホートに基づく研究に従う方法多重遺伝子型を示します。

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

candidate-gene-testing-clinical-cohort-studies-with-multiplexed

Universidad San Sebastian

Research

JoVE Journal

Genetics

10.0K ビュー.  Max-Delbrück Center for Molecular Medicine. 複雑な人間の病気に貢献する遺伝的変異の同定機構を識別することができます。ここでは、候補者の遺伝子または遺伝子パスウェイ解析低コストでカバレッジを最大化、コホートに基づく研究に従う方法多重遺伝子型を示します。

ビデオ: Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

新たに単離したヒト絨毛細胞栄養芽層に対するsiRNAトランスフェクションおよびEMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

遺伝学

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.
Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.
The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

のサブタイピング<em&gt;カンピロバクター・ジェジュニ</em&gt; SSP。<em&gt; doylei</em&gt;質量分析ベースのPhyloProteomics（MSPP）を使用して分離株

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

液体クロマトグラフィー/質量分析法による有毒フグの改善されたポリメラーゼ連鎖反応 - 制限断片長多型ジェノタイピング

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

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.

ヒト初代細胞培養物における概日時計遺伝子発現やホルモン分泌のパラレル測定

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

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.

過敏性腸症候群におけるmiRNA循環の摂動は多重化ハイスループット遺伝子発現プラットフォームを使用して検出します

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

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

隠すビートルにノックダウン飼育し、二本鎖RNA媒介遺伝子、<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

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.

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.

内部標準でFACSおよび定量PCRを用いた単一細胞遺伝子発現プロファイリング

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

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

補体受容体1長多型のための高分解能Melting PCR Genotyping：アルツハイマー病の遺伝子感受性評価のための革新的なツール

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

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.

遺伝子破壊ミュータンス連鎖球菌緊張せず遺伝子クローニングの生成

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.

スループットを向上させると衝撃されたオオムギ糊粉層における遺伝子発現を測定

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