이 연구는 국민 건강과 호주의 의료 연구위원회 (프로젝트 부여 안돼. 6,288,480)와 스코비와 클레어 맥키논 트러스트에 의해 지원되었다.

1. RT 반응을 Micromixing <ol><li> micromixing과 RT 반응을 수행하기 전에, RT 반응의 원하는 온도로 micromixer를 평형. <ol><li> 37 내부 micromixer를 놓고 ° C (또는 RT 공급 업체에서 권장하는 온도) RT 반응의 시작하기 전에 적어도 1 시간 보육. </li></ol></li><li> 반대 transcriptase 공급자에 따라 RT 믹스를 설정 (Promega, Qiagen에서 Omniscript에서 예 MMTV - RT) 방법 대신 NG - μg의 총 RNA 입력 (예 : 0.1-1pg/μl)의 사용 단일 셀 금액 제외 금액은 공급 업체에 의해 권장합니다. <ol><li> 우리는 표준 살균, nuclease없는 200μL 얇은 벽의 PCR 튜브를 사용합니다. </li></ol></li><li> 그들은 혼합 준비 마자 안전 micromixer에 위치 RT 튜브. micromixer은 RT 반응의 기간 동안 37 ° C 배양기 안에서 유지됩니다. </li><li> 적절한 빈도와 micromixing의 진폭을 선택합니다. 이 예제에서 우리는 150Hz 사용합니다. </li><li> 에 micromixer 스위치 및 RT 반응 (60 분)의 전체 기간 동안 그것을 두십시오. </li><li> micromixer 끄고 RT 반응이 완료되면 RT 튜브를 꺼내. </li><li> 얼음 RT 튜브를 삽입 및 정량 중합 효소의 연쇄 반응 (qPCR 또는 실시간 PCR)과 같은 추가 애플 리케이션을 정상적으로 진행합니다. </li></ol> 2. 대표 결과 : RT 반응의 맥락에서 micromixing의 장점은 다른 혼합 방법이 (예를 들어, 흔들림 vortexing 또는 분쇄) 사용하는 경우에 상대적으로 cDNA 수확량의 증가이다. 이것은 cDNA를 사용 정량 중합 효소의 연쇄 반응 (qPCR 또는 실시간 PCR)의 후속 분석에 따라, 예를 들어 볼 수 있습니다. 그림 2A 바로 전에 RT 보육 (흑백, &quot;분쇄&quot;로 RT 반응의 분쇄와 비교하여 처음 5 분 동안 micromixing의 효과 (파란색, &quot;micromixed5&quot;) 또는 전체 60 분 (빨간색, &quot;micromixed60&quot;)를 보여줍니다 ). 이 경우에는 앞의 RT 반응의 부피 25μL이고 그것은 총 RNA의 2.5pg이 포함되어 있습니다. cDNA 대표 Nurr - 1 mRNA를 검출하기위한 primers를 사용하여 qPCR의 흔적은 위에 표시된되며, 수단 ± 같은 실험을 3 반복으로 50 %의 최대 형광에 도달 사이클의 수를 전문가, 대리인은 아래 꾸몄다 있습니다. 별표는 통계적으로 중요한 차이 (Tukey 다중 비교와 편도 ANOVA)을 나타냅니다. 앞의 RT 반응의 전체 60 분 micromixing은 분쇄에 비해 상당한 개선을 생산하는 반면 위의 RT 반응의 첫 번째 5 분 micromixing은 분쇄 이상이 아닌 상당한 개선을 생산합니다, 규모있는 것은 약 15 qPCR에 해당되었다 주기, 평균. micromixing의 혜택도 얻을 이후 qPCR 제품의 곡선 분석을 용해에서 볼 수 있습니다. 유일한 일관성 qPCR을 생산 처음 5 분 또는 분쇄를위한 micromixing 반면에 이전 RT 반응의 전체 60 분 micromixing 그림 2B에 설명된 실험, 중복 표본의 일관성 qPCR 신호 (그림 2Ba, 2 빨간 흔적) 결과 흔적 (그림 2Ba, 2 블루 흔적 2 검은 흔적, 각각). 회색 흔적은 qPCR 반응 밖으로 버려졌습니다 cDNA있는 부정적인 컨트롤 ( &quot;neg.&quot;)입니다. 본 실험에서 qPCR 제품은 확실했다 그들의 온도 (즉, 용해 곡선 분석)을 올렸에 의해 변성되었을 때 그 적절한 PCR 제품 (즉, mRNA의보다 높은 농도는 반대로 qPCR에 의해 베꼈는데 및 증폭했을 때 얻은 동일했습니다 amplicon, 표시되지 데이터) 60 분 (그림 2Bb, 2 붉은 자취)에 대한 micromixed했다만이 다음과 같은 RT 반응을 제시했다. 모든 다른 (즉 micromixed5, 분쇄 및 부정적인 컨트롤)은 대체 amplicon 제품 (예 : 뇌관의 dimers) 생성. <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="그림 1"/> 그림 1. 표준 실험실 RT 반응에 micromixing를 적용하기위한 프로토콜의 흐름 다이어그램. 물리적인 원리 참여, micromixer 및 내부 micromixer 구축에 더 자세한 설명은 참고 문헌 13,14에서 제공하는 <img src="/files/ftp_upload/3144/3144fig2.jpg" alt="그림 2"/> 그림 2. 달리 표준 실험실 RT 반응에서 mRNA의 단일 셀 수량 구성된 RT 반응 앞에 적용 micromixing의 장점을 보여주는 대표 qPCR 데이터입니다. Nurr - 1 유전자를 인코딩 cDNA 나타내는 mRNA를 검출 qPCR 추적 예. 이전 RT 반응이 25μL의 볼륨 총 RNA의 2.5pg 포함되어 ° C 60 분 37 인큐베이터에서 수행되었다. RT 반응은 R의 분쇄에 의해 혼합되었다T는 이전 (빨간색 흔적, &quot;micromixed60&quot;RT 반응 (푸른 흔적 &quot;micromixed5&quot;)의 처음 5 분 동안 micromixed 또는 RT 반응의 전체 60 분 micromixed 부화 (검은 흔적, &quot;분쇄&quot;)에 혼합 ). 수단 ± 50 % 최대 형광에 도달하는 데 필요한 qPCR 사이클의 수를 전문가, 대리인은 N은 아래 꾸몄다있는이 실험 = 3 반복합니다. 별표 상당한 차이 (Tukey 다중 비교와 단방향 ANOVA)을 나타냅니다. B 다른 qPCR 실험이 25μL의 볼륨 총 RNA의 25pg를 포함하는 RT 반응에 따라 이번 Nurr - 1 cDNA 제품을 검토. qPCR의 흔적 (A)뿐만 아니라 얻은 qPCR 제품의 곡선 분석을 용해가 (B) 다른 혼합 조건 표시됩니다. 아무 cDNA가 이전 RT 반응에 포함되지 않은있는 부정적인 컨트롤도 (회색 흔적 &quot;neg.&quot;) 포함되어 있습니다. mRNA의보다 높은 농도로 RT 반응도 수행 및 (b) micromixed60에 대한 (붉은 흔적) 샘플 사람들에게 동일한 용해 곡선 봉우리를 (데이터가 표시되지 않음) 생산되었다.

<ol>
	<li>Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Funct+Genomic+Proteomic.">Brief Funct Genomic Proteomic.</a> 2 (1), 31-31 (2003).</li><li>Aumann, T. D., Gantois, I., Egan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Exp Neurol. 213 (2), 419-419 (2008).</li><li>Ottino, J. M., Wiggins, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Philos Transact A Math Phys Eng Sci. 362 (1818), 923-923 (2004).</li><li>Losey, M. W., Jackman, R. J., Firebaugh, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Microelectromech. Syst. 11, 709-709 (2002).</li><li>Bontoux, N., Dauphinot, L., Vitalis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lab Chip. 8 (3), 443-443 (2008).</li><li>Marcus, J. S., Anderson, W. F., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 78 (9), 3084-3084 (2006).</li><li>Marcus, J. S., Anderson, W. F., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 78 (3), 956-956 (2006).</li><li>Warren, L., Bryder, D., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc Natl Acad Sci U S A. 103 (47), 17807-17807 (2006).</li><li>Jiao, Z. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 6, 847-847 (2009).</li><li>Ahmed, D., Xiaole, M., Juluri, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 7, 727-727 (2009).</li><li>Paxton, W. F., O'Hara, M. J., Peper, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 80, 4070-4070 (2008).</li><li>Autom, L. a. b. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 11, 399-399 (2006).</li><li>Petkovic-Duran, K., Manasseh, R., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 47 (4), 827-827 (2009).</li><li>Boon, W. C., Petkovic-Duran, K., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 50 (2), 116-116 (2011).</li><li>Liu, R. H., Lenigk, R., Druyor-Sanchez, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 75 (8), 1911-1911 (1911).</li><li>Riley, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ann+Rev+Fluid+Mech.">Ann Rev Fluid Mech.</a> 33, 43-43 (2001).</li><li>Whitehill, J., Neild, A., Ng, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applied Physics Letters. 96 (5), 053501-053501 (2010).</li></ol>

저자는 공개 없어요.

표준 실험실 RT 반응에 micromixing의 신청 방법은 물론, 참여 할 수 여기에 설명된 모든 방법 (예 : 세포 용해, 레이저 캡처 microdissection)을 통해 수확 mRNA. 그것은 또한 모든 브랜드 또는 형식 RT 시약의 어떤 온도 (micromixer의 재료의 허용 오차 이내), 시간의 기간을 구성하실 수 있습니다. 예를 들어, 우리는 무작위로 hexamer 또는 oligo - DT primers로 구성된 RT 반응에서 개선 cDNA 수율을 관찰했습니다. 그것은 ...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 총 RNA는 스냅 언 심하게 준비 성인 마우스 midbrain의 조각으로부터 격리되었다 </td><td></td><td></td><td></td></tr><tr><td> PicoPure RNA 격리 키트 </td><td> 아크 투 르스, CA, 미국 </td><td> KIT0204 </td><td> 키트는 현재 응용 Biosystems에서 구할 수 있습니다 </td></tr><tr><td> DNA - 무료 DNase 처리 및 제거 시약 </td><td> 앰비온 </td><td> AM1906M </td><td></td></tr><tr><td> 랜덤 hexamer primers </td><td> Promega </td><td> C1181 </td><td></td></tr><tr><td> AMV - RT </td><td> Promega </td><td> M5101 </td><td></td></tr><tr><td> dNTP 세트 </td><td> Promega </td><td> U1240 </td><td></td></tr><tr><td> RNasin Ribonuclease 억제제 </td><td> Promega </td><td> N2111 </td><td></td></tr><tr><td> Nuclease - 프리 워터 </td><td> Promega </td><td> P1193 </td><td></td></tr><tr><td> SYBR 그린 PCR 마스터 믹스 </td><td> 응용 Biosystem </td><td> 4,309,155 </td><td></td></tr><tr><td> Hprt 전달 (20mer) : CTT TGC TGA CCT GCT GGA TT </td><td></td><td></td><td></td></tr><tr><td> Hprt 반대 (20mer) : 문신 GTC CCC CGT TGA CTG AT </td><td></td><td></td><td></td></tr><tr><td> Nurr1 전달 (23mer) : 편대의 편대 장 CTC CGA CTT TTT AAC TCC AG </td><td></td><td></td><td></td></tr><tr><td> Nurr1 반대 (23mer) : GGT 개그 GTC CAT GCT AAA CTT GA </td><td></td><td></td><td></td></tr><tr><td> 1,000 분광을 NanoDrop. </td><td> 써모 과학 </td><td></td><td></td></tr><tr><td> 코벳 로터 진 RG - 6000 </td><td> 코벳 생명 과학 </td><td></td><td> 코벳 생명 과학 2008 년 7 월 QIAGEN에 의해​​ 인수되었다 </td></tr></tbody></table>

increasing cdna yields from single-cell quantities of mrna in standard laboratory reverse transcriptase reactions using acoustic microstreaming

세포의 행동과 연관 유전자 발현은 이상적으로 단일 세포 수준에서 이루어집니다. 그것이 베꼈는데를 반대하기 전에 하나의 셀 (1월 1일에서 5일까지 - 50pg 총 RNA의 % 또는 0.01 - 2.5pg mRNA, 셀 하나 당)에있는 불안 정한 mRNA의 작은 금액은 대부분이 저하하기 때문에 그러나, 이것은 쉽게 달성되지 않습니다 안정적인 cDNA 사본으로. 예를 들어, 표준 실험실 시약 및 하드웨어를 사용하여, 유전자의 단지 소수가 질적으로 전지 2마다 평가하실 수 있습니다. mRNA의 단일 셀 금액 구성된 표준 실험실 반전 transcriptase (RT) 반응 (microliter 볼륨에서 즉, 표준 시약)의 효율성을 높이는 한 가지 방법은 그것이 저하되기 전에 mRNA가 cDNA로 변환 수 있도록보다 신속하게 시약을 혼합하는 것입니다. 그러나 microliter 비늘에서 액체 흐름이 층류이기 때문에 이것은 하찮은되지 않습니다, 즉 혼합의 현재 방법 (흔들림 vortexing 및 분쇄 즉)은 효과적으로 시약을 혼합하는 데 충분한 혼란 움직임을 생산하기 위해 실패합니다. 이 문제를 해결하기 위해서, 마이크로 스케일 혼합 기법 3,4을 사용해야합니다. microfluidic 기반의 혼합 기술의 숫자가 성공적으로 RT 반응 수율에게 5-8를 증가하는 개발되었습니다. 그러나, microfluidics 기술은 상대적으로 비싸고 아직 널리 사용할 수없는 전문적인 하드웨어가 필요합니다. 저렴, 더 편리한 솔루션은 바람직합니다. 본 연구의 주요 목적은 mRNA의 단일 셀 수량 구성된 표준 실험실 RT 반응에 소설 &quot;micromixing&quot;기술의 응용은 크게 자신의 cDNA 수확량을 증가하는 방법을 보여줍니다하는 것입니다. 우리는 cDNA 산출이 가능하게 약 10 - 100 - 배로 증가 발견 (1) 세포마다 분석하는 유전자의 큰 숫자, 유전자 발현 (2) 더 많은 정량 분석​​하고, 낮은 풍부한 유전자 (3) 좋은 검출 단일 세포 인치 micromixing는 음향은 9-12 작은 장애물 주위에 전파 음파가 장애물 근처에 의미 흐름을 만드는 현상을 microstreaming을 기반으로합니다. 우리는 주요 단순화와 음향 microstreaming 기반 장치 (이하 &quot;micromixer&quot;) 개발, 음향 microstreaming는 시스템이 굴곡 13 작은 반경을 가진 액체 공기 인터페이스를함으로써 오디오 주파수에서 얻을 수 있습니다. 튜브 솔루션 microliter 볼륨의 초승달 모양은 곡률 적절하게 작은 반경을 제공합니다. 오디오 주파수의 사용은 하드웨어가 저렴 13 다용도 수있다는 것을 의미하고 표준 연구소 sonicators 함께 할 수처럼 핵산 및 다른 생화 학적 시약이 손상되지 않습니다.

Watch this Scientific Journal Video about Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming at JoVE.com

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

우리는 달리 일반 실험실 역방향 전사 반응에서 mRNA의 단일 셀 수량로부터 cDNA 수율을 높이기 위해 새로운 방법을 설명합니다. 참신은 흔들림 vortexing 또는 분쇄보다 효과적으로 microliter 비늘에서 유체를 섞어, 음향 microstreaming의 현상을 활용 micromixer의 사용에 있습니다.

어쿠스틱 Microstreaming를 사용하여 표준 실험실 역방향 Transcriptase 반응에서 mRNA의 단일 세포로부터 cDNA 수량 산출 증가

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

increasing-cdna-yields-from-single-cell-quantities-mrna-standard

Research

JoVE Journal

Bioengineering

10.1K 조회수.  University of Melbourne. 우리는 달리 일반 실험실 역방향 전사 반응에서 mRNA의 단일 셀 수량로부터 cDNA 수율을 높이기 위해 새로운 방법을 설명합니다. 참신은 흔들림 vortexing 또는 분쇄보다 효과적으로 microliter 비늘에서 유체를 섞어, 음향 microstreaming의 현상을 활용 micromixer의 사용에 있습니다.

비디오: Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

일반 잉크젯 프린터를 사용하여 과도 세포막의 세공 만들기

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

연구

생체공학

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

단일 세포 분비물의 공간 - 시간 영상의 레이블이없는 기술

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

단일 셀 게놈 시퀀싱 초고 처리량 미세 플랫폼

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects, are often difficult to provide for further studies or third parties due to the consumptive nature of the samples. Although now widely distributed over the Pacific coasts of Asia, Australia and North America, individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries. In this article, we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries. These libraries were then compared to the publicly available oyster genome, and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I (COX1) and NADH Dehydrogenase (ND). The suitability of the generated cDNA library is also demonstrated by cloning and expression of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS), which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.

We describe a strategy for how to use RNA samples from unreferenced Pacific oyster specimens, and evaluate the genetic material by comparison with publicly available genome data to generate a virtually sequenced cDNA library.

참조되지 않은 태평양 굴에서 사실상 순차적 cDNA 라이브러리 생성을 위한 수렴 전략

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

생물학적 활성 Coralline Matrix를 사용한 해리 된 신경 세포 배양의 내구성 향상

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

음향 분리기사용 CHO 수확세포 배양액의 1차 해명

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

목재 접착제의 응집력 개발을 위한 표준 시험 방법 ASTM D 7998-19

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

자기 고열증 및 포토열 조합 암 치료를 위한 자기, 음향 및 광학 삼중 반응 마이크로 버블

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

인간 유도 만능 줄기 세포 유래 심근 세포에서 단세포 광학 작용 잠재적 측정

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

마이크로 패턴화된 엘라스토머를 이용한 단일 세포 수축성의 단순화된 고처리량 분석

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...