저자 cellulolytic 효소의 선물 Viksø - 닐슨 (Novozymes)을 감사드립니다. 이 작품은 FP7 갱신으로하고 BBSRC 프로젝트 BB/G016178 및 BB/G016194에 의해 후원되었다.

1. 샘플 준비 우리는 일반적으로 풀의이나 우디 식물 중 하나에서 비롯된와 함께 작동합니다. 풀의 재료의 경우 우리는 성숙에 식물을 성장하고, 노화가 완료되면 종자 개발은 우리가 마른 줄기를 수집합니다. 줄기는 4mm 세그먼트로 썰어 세 밀링 공 2 ML 튜브에 배치됩니다. 우디 자료의 경우, 샘플은 물론 나무 파일을 거친 톱밥을 접지하고 다음 추가 처리를 위해 공을 공장에 배치. 샘플은 연삭 / 역 (그림 1) 포맷 내에 선반에 배치됩니다. 이 기지는 순차적으로, grinds 믹스, 각 샘플의 무게. 샘플마다 필요한 반복의 수는 특정 입자 크기에 대한 본질적인 다양성이 결정되는 예비 실험의 일련의 지상 자료를 테스트하여 설정됩니다. 2 ML의 튜브는 하단에 피어싱하고 자료 선택된 잘 위에 유리병의 진동에 의해 적절합니다. 진동 팔은 분말의 정확한 분배에 대한 허용의 균형에서 피드백에 의해 제어됩니다. 로봇 표준 분석 / 음 4 MG를 dispenses 4 반복 대부분의 경우에 필요합니다. 이것은 20 식물 샘플 / 플레이트가 분석 수 있습니다. 2. 전처리 샘플 서식되면, 96 - 웰 플레이트는 자동 액체 처리 시스템 (그림 2)에 의해 처리됩니다. 여기 가벼운 전처리가 산성이나 알칼리성 솔루션을 350 μL를 추가하고 적응 블록에 접시를 가열하여 식물 재료에서 수행됩니다. 분석하는 동안 증발을 방지하려면 96 잘 플레이트는 실리콘 매트와 함께 봉인하고 있습니다. 전처리의 온도와 시간은 연구되고있는 재료에 따라 수정할 수 있습니다. 최대 온도는하지만, 100 ° C.입니다 엄격한 pretreatments 테스트를위한 대안 절차는 온라인에서 자료를 pretreat 및 프로세스에서 pretreatmet을 제거하는 것입니다. 3. 가수 분해 <ol><li> 자료 pretreated 후, 로봇이 자동으로 전처리 솔루션, 그리고 세척, 모든 우물, 850 μL 25 MM 아세테이트 버퍼 5 번 제거합니다. rinses은 전처리 솔루션에 버퍼를 추가하고, 이후 예제에서 고체가 정착 있도록 이후 전체 볼륨의 50 %를 제거하여 실시하고 있습니다. 흡인의 높이는 우물의 바닥에서 고체를 aspirating 피하기 위해 액체 질량의 절반에서 설정되며이 방법을 사용하여 고체의 손실이 무시할 수 있습니다. 이들은 세척 후 잘의 산도는 4.5이며, 자료 효소 가수 분해를위한 준비가되었습니다. </li><li> cellulases 및 hemicellulases의 솔루션을 포함하는 효소 혼합물은 로봇에 의해 추가됩니다. 각 우물에, 효소 혼합물의 850 μL이 총동원되고 번호판이 50 ° C 떨고 인큐베이터로 이동합니다. 표준 가수 분해는 8 H에 대해 수행하지만, 이번 실험의 목적에 적용할 수 있습니다. 풀밭에 사용되는 표준 효소 로딩은 자료 7 FPU / g이다. </li></ol> 4. 설탕 줄이는 감지 가수 분해 이후에 릴리스된 설탕 감소의 결정은 수정 3 - 메틸 - 2 - benzothiazolinone hydrozone (MTBH) 방법 3을 사용하여 수행되었습니다. 그것은 자동화와 같은 쉬운 단백질과 같은 성분의 간섭에 대한 이상 감염될 때문에 MBTH가 가장 적합한 방법으로 선정되었습니다. 그것이 정확하게 바이오 매스 hydrolysates에있는 농도에서 설탕을 계량 수 있도록 표준 광학 96 잘 플레이트에 적합합니다 250 μL의 최종 볼륨과 로봇 플랫폼에 사용하기 위해이 매우 민감한 방법을 수정했습니다. 아래의 모든 단계를 자동으로 수행되고 절감 설탕 세 가지 독립적인 결정은 각 당화 반응을 위해 실시되었다. <ol><li> hydrolyzate 30 μL은 깊은 우물을 판에서 가져온와 skirted PCR 96 자 판 25 MM의 아세트산 나트륨 버퍼 45 μL와 혼합되었다. </li><li> 1N NaOH 25 μL 및 0.43 MG / ML MBTH 및 0.14 MG / ML DTT을 포함하는 솔루션을 50 μl가 추가되었습니다. </li><li> 혼합 후, PCR 플레이트는 60 ° 온수했다 모든 96 우물에 열을의 정확히 같은 금액을 제공하는 thermocycler 또는 이와 유사한 장치 20 분 C. </li><li> 그 결과 반응은 광학 판으로 전달했다. </li><li> oxidising 시약 (0.2 % FeNH4 (SO4) 2, 0.2 % sulfamic 산성과 0.1 % HCL) 100 μl를 추가합니다. 최소한 1 H. 위해 잘 혼합하고 개발하기 위해 떠나 </li><li> 각 플레이트는 50 nmol, 100 nmol 150 nmol 포도당의 표준 반응을 포함해야합니다. 광학 접시는 620 nm의에서 읽을 수 있습니다. </li></ol> 5. 대표 결과 : 분석의 종류의 예는 그림 3과 4로 표시됩니다. 모든 결과는 자동으로 플랫폼을 사용하여 얻은되었습니다. 그림 3을 다시cellulolytic 효소의 증가 금액과 지상 포플러에서 8 H의 incubations에 의해 발표 설탕을 감소에서 증가를 제공합니다. 그림 4는 산성 및 포플러 샘플 알칼리 전처리의 효과를 제공합니다. NaOH의 전처리가보다 효율적입니다 가수 분해 동안 당분의 석방을 촉진에 희석 H2SO4 전처리. 가수 분해 후 측정 당분의 금액은 전처리가 1M 이상의 NaOH의 농도를 사용하여 수행할 때 줄어 듭니다. <img src="/files/ftp_upload/3240/3240fig1.jpg" alt="그림 1" /> 그림 1. 분쇄 및 바이오 매스의 96 음 형식에 대한 로봇 역 <img src="/files/ftp_upload/3240/3240fig2.jpg" alt="그림 2" /> 그림 2. 높은 처리량 당화 분석을위한 액체 취급 역 <img src="/files/ftp_upload/3240/3240fig3.jpg" alt="그림 3" /> 그림 3. 포플러 샘플에서 설탕에 해당하는 금액을 줄이는 릴리스에 다른 효소 loadings 효과 <img src="/files/ftp_upload/3240/3240fig4.jpg" alt="그림 4" /> 산성 전처리보다 같은 온도와 시간에 NaOH의 pretreatments 이후에 출시된 90에서 H2SO4의 다른 비율로 pretreament ° 30 분 C 이후에 릴리스된 그림 4. 포플러 샘플 당화에 다른 pretreatments의 효력. A.의 슈거스. B.의 슈거스 .

<ol>
	<li>Carpita, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Biogenesis+of+the+Cell+Walls+of+Grasses.">Structure and Biogenesis of the Cell Walls of Grasses.</a> Annu Rev Plant Physiol Plant Mol Biol. 47, 445-476 (1996).</li><li>Gomez, L. D., Steele-King, C. G., McQueen-Mason, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustainable+liquid+biofuels+from+biomass:+the+writing's+on+the+walls.">Sustainable liquid biofuels from biomass: the writing's on the walls.</a> New Phytol. 178, 473-485 (2008).</li><li>Anthon, G. E., Barrett, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+reducing+sugars+with+3-methyl-2-benzothiazolinonehydrazone.">Determination of reducing sugars with 3-methyl-2-benzothiazolinonehydrazone.</a> Anal Biochem. 305, 287-289 (2002).</li><li>Gomez, L. D., Whitehead, C., Barakate, A., Halpin, C., McQueen-Mason, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+saccharification+assay+for+determination+of+digestibility+in+plant+materials.">Automated saccharification assay for determination of digestibility in plant materials.</a> Biotechnol Biofuels. 3, 23-23 (2010).</li></ol>

Variations of the standard saccharification protocol can be used in the same platform for determining the activity of cellulolytic enzymes (i.e. by variation of the enzyme concentrations used in a plate as well as using paper as substrate); comparison of the efficiency of several enzyme mixtures on a specific material; time course for saccharification; etc.
A standard saccharification protocol to compare the saccharification potential in different plant materials involves an eight hour hydrolysis (Figures 3 and 4). Most of the plant materials analysed requires four replicates. Under these conditions, the platform can process 80 samples/day. This analysis is being used to screen large populations of barley, maize, and brachypodium in order to establish variability in saccharification potential and the genes involved in its determination4.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 로봇을 연삭 및 무게 </td><td> Labman 자동화 </td><td></td></tr><tr><td> 모자 2 ML 마이크로 튜브 </td><td> Sarstedt (주) </td><td> 72.694 </td></tr><tr><td> 5mm 스테인리스 비즈 </td><td> Qiagen (주) </td><td> 69,989 </td></tr><tr><td> 1.2ml Abgene 스퀘어 웰 저장 플레이트 </td><td> 피셔 과학 (주) </td><td> TUL - 050 - 050C </td></tr><tr><td> 96 스퀘어 웰 플레이트에 대한 왓먼 캡 매트 </td><td> 피셔 과학 (주) </td><td> 7704-0104 </td></tr><tr><td> 액체 hanling 로봇 </td><td> Tecan 그룹 (주) </td><td> 자유 에보 (Evo) 200 </td></tr><tr><td> Sulphuric 수 소산 </td><td> 피셔 과학 (주) </td><td> S/9231/PB17 </td></tr><tr><td> 수산화 나트륨 </td><td> 피셔 과학 (주) </td><td> BPE359 - 500 </td></tr><tr><td> 아세트산 나트륨 </td><td> 시그마 - 알드리치 </td><td> S8750 - 500G </td></tr><tr><td> 초산 </td><td> 피셔 과학 (주) </td><td> A/0420/PB17 </td></tr><tr><td> Novozyme 188 </td><td> Novozyme </td><td> DCN00214 </td></tr><tr><td> Celluclast 1.5L </td><td> Novozyme </td><td> CCN03122 </td></tr><tr><td> 96 잘 PCR 전체 스커트 플레이트 </td><td> Sarstedt (주) </td><td> 72.1980.202 </td></tr><tr><td> D - 포도당 </td><td> 피셔 과학 (주) </td><td> G/0450/53 </td></tr><tr><td> 3 - 메틸 - 2 - benzothiazolinone hydrazone 하이드로 클로라이드 하이드 레이트 </td><td> 시그마 - 알드리치 </td><td> 129739 - 25G </td></tr><tr><td> DL - dithiothreitol </td><td> 시그마 - 알드리치 </td><td> D9163 - 1G </td></tr><tr><td> 코닝 96 microplate 잘 플랫 바텀 </td><td> 피셔 과학 (주) </td><td> TKT - 521 - 050H </td></tr><tr><td> 암모늄 철 (III) 황산의 dodecahydrate </td><td> 시그마 - 알드리치 </td><td> F1668 - 250G </td></tr><tr><td> Sulfamic 수 소산 </td><td> 시그마 - 알드리치 </td><td> 242772 - 500G </td></tr><tr><td> 염산 </td><td> 피셔 과학 (주) </td><td> 12462-0026 </td></tr></tbody></table>

high-throughput saccharification assay for lignocellulosic materials

공장 리그노셀룰로오스성 바이오 매스를 만드는 다당류는 이후 biorefinery를 구축에서 사용할 수있는 당분의 범위를 생산하는 세분 수 있습니다. 이 원료는 화석 연료에 대한 현재 종속성을 대체하기 위해 지속 가능하고 탄소 중립 모두는 새로운 산업 플랫폼을, 구성 것입니다. 리그노셀룰로오스성 자료에 관찰 해체하는 말을 안들음은 식물 세포 벽 몇 가지 본질적인 특성에 의해 생산됩니다. 결정 셀룰로오스는 그러한 xylans 및 arabinoxylans 같은 매트릭스 다당류에 포함된하고, 전체 구조는 1 소화도 어려운 페놀 수지 carbon 폴리머 리그닌에 의해 쌌다입니다. 우리가 당화 2의 다양성을 평가하는 주요 세포 벽의 당화에 대한 병목 현상 또한 화면 돌연변이 및 번식 인구를 발견할 필요가 식물 재료의 소화율을 개선하기 위해. 이러한 작업은 높은 처리량 접근을 필요로하고 여기에 우리는 96 - 웰 플레이트 형식으로 당화 분석을 수행할 수 분석 플랫폼을 제시. 이 플랫폼은 다양한 식물 종 대형 인구의 lignocellulose의 소화율의 심사를 허용하도록 개발되었습니다. 우리는 많은 번호가 자동으로 시스템에 신속하게 평가하도록, 부드러운 전처리, 부분 가수 분해 효소와 설탕의 결정에 대한 반응 볼륨을 축소했습니다. 이것은 자동화된 플랫폼은 재현성 방식으로 표준 입자 크기로 공장 자재를 줄이기 위해 통제 조건 하에서 볼 밀링을 수행, 바이오 매스의 양을 밀리그램와 함께 작동합니다. 샘플 지상되면, 자동 서식 로봇 96 딥 웰 플레이트 (그림 1)의 해당 우물에 물질의 지정 및 기록된 금액을 dispenses. 일반적으로, 우리는 4 분석을 위해 복제가 4 우물에 동일한 자료를 분배. 접시가 원하는 레이아웃에있는 식물로 가득되면, 그들은 수동으로 액체 처리 스테이션 (그림 2)로 이동됩니다. 이 기지에서 샘플은 희석 염산 알칼리 중 하나와 함께 가벼운 전처리의 대상이되며 90 ° C. 최대 온도에서 incubated 전처리 솔루션은 연속적으로 제거되고 샘플은 가수 분해에 적합한 산도에게 반환하는 버퍼로 씻어서 있습니다. 샘플은 다음 50 시간 가변 길이에 대한 효소 혼합 ° C.와 incubated 아르 나누어지는는 hydrolyzate에서 가져온되고 줄이고 설탕이 자동으로 MBTH colorimetric 방법에 따라 결정됩니다.

Watch this Scientific Journal Video about High-throughput Saccharification Assay for Lignocellulosic Materials at JoVE.com

High-throughput Saccharification Assay for Lignocellulosic Materials

식물 바이오 매스 샘플을 다수의 당화 잠재력을 결정하는 간단하고 빠른 방법을 설명합니다. 이 분석을 위해 자동화된 플랫폼은 96 접시와 잘 풀어 설탕의 전처리, 가수 분해 및 부량의 후속 성능 분석을위한 식물 바이오 매스의 준비를 포함한다.

리그노셀룰로오스성 자료에 대한 높은 처리량 당화 분석

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

high-throughput-saccharification-assay-for-lignocellulosic-materials

Research

JoVE Journal

Biology

13.2K 조회수.  University of York. 식물 바이오 매스 샘플을 다수의 당화 잠재력을 결정하는 간단하고 빠른 방법을 설명합니다. 이 분석을 위해 자동화된 플랫폼은 96 접시와 잘 풀어 설탕의 전처리, 가수 분해 및 부량의 후속 성능 분석을위한 식물 바이오 매스의 준비를 포함한다.

비디오: High-throughput Saccharification Assay for Lignocellulosic Materials

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

높은 처리량 SHAPE를 사용하여 RNA 이차 구조 예측

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

연구

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High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

펩티드 에피토프의 정량 분석​​을위한 높은 처리량 MHC II 결합 분석

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

루시 페라 제 발현 재조합 바이러스의 높은 처리량의 적정

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

3&#39;LIFE의 분석을 사용하여 높은 처리량 miRNA의 표적의 탐지

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

높은 처리량<em&gt; 다니오 레 리오 (rerio)</em&gt; 에너지 지출 분석

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.