The beginning of the end for microarrays?pp585 - 587 Jay Shendure doi:10.1038/nmeth0708-585
Two complementary approaches, both using next-generation sequencing, have successfully tackled the scale and the complexity of mammalian transcriptomes, at once revealing unprecedented detail and allowing better quantification.
Strategies for the comprehensive identification of transcript isoforms produced from specific genomic loci make use of and expand existing tools and resources.
Microfluidics: streamlining discovery in worm biologypp589 - 590 S Elizabeth Hulme, Sergey S Shevkoplyas & Aravinthan Samuel doi:10.1038/nmeth0708-589
Advances in the application of microfluidics technology to biological assays using the model organism Caenorhabditis elegans help to automate otherwise time-consuming experiments.
Whispering-gallery-mode biosensing: label-free detection down to single moleculespp591 - 596 Frank Vollmer & Stephen Arnold Published online: 27 June 2008|doi:10.1038/nmeth.1221 Abstract|Full text|PDF
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Isoform discovery by targeted cloning, 'deep-well' pooling and parallel sequencingpp597 - 600 Kourosh Salehi-Ashtiani, Xinping Yang, Adnan Derti, Weidong Tian, Tong Hao, Chenwei Lin, Kathryn Makowski, Lei Shen, Ryan R Murray, David Szeto, Nadeem Tusneem, Douglas R Smith, Michael E Cusick, David E Hill, Frederick P Roth & Marc Vidal Published online: 15 June 2008|doi:10.1038/nmeth.1224
The complete set of coding sequences, including all splice isoforms, is not known for any metazoan organism. Combination of a normalized pooling scheme and a new assembly algorithm with 454 sequencing yields a methodological pipeline for isoform discovery. The validated pipeline may now be applied genome-wide.
Transgenesis via permanent integration of genes in repopulating spermatogonial cells in vivopp601 - 603 Suveera Dhup & Subeer S Majumdar Published online: 15 June 2008|doi:10.1038/nmeth.1225
Conventional techniques for generating transgenic mice are quite costly, require substantial resources and necessitate killing the mouse. In contrast, in vivo electroporation of repopulating spermatogonial cells in the mouse testis can produce male mice for siring multiple distinctive transgenic founders for over a year.
Lifeact: a versatile marker to visualize F-actinpp605 - 607 Julia Riedl, Alvaro H Crevenna, Kai Kessenbrock, Jerry Haochen Yu, Dorothee Neukirchen, Michal Bista, Frank Bradke, Dieter Jenne, Tad A Holak, Zena Werb, Michael Sixt & Roland Wedlich-Soldner Published online: 08 June 2008|doi:10.1038/nmeth.1220
Current approaches for live imaging of cellular actin dynamics have several drawbacks. Now the use of Lifeact, a 17-aa actin-binding peptide from yeast that is not present in higher eukaryotes, allows imaging of actin dynamics in live mammalian cells without disruption of function and without competition with endogenous binding proteins.
In vitro whole-organ imaging: 4D quantification of growing mouse limb budspp609 - 612 Marit J Boot, C Henrik Westerberg, Juanjo Sanz-Ezquerro, James Cotterell, Ronen Schweitzer, Miguel Torres & James Sharpe Published online: 30 May 2008|doi:10.1038/nmeth.1219
A combination of improved in vitro embryo culture and optical projection tomography allows development of the mouse limb bud to be monitored over time. Developmental changes seen in vitro are benchmarked against in vivo development, and tissue movements are quantitatively described.
Stem cell transcriptome profiling via massive-scale mRNA sequencingpp613 - 619 Nicole Cloonan, Alistair R R Forrest, Gabriel Kolle, Brooke B A Gardiner, Geoffrey J Faulkner, Mellissa K Brown, Darrin F Taylor, Anita L Steptoe, Shivangi Wani, Graeme Bethel, Alan J Robertson, Andrew C Perkins, Stephen J Bruce, Clarence C Lee, Swati S Ranade, Heather E Peckham, Jonathan M Manning, Kevin J McKernan & Sean M Grimmond Published online: 30 May 2008|doi:10.1038/nmeth.1223
Application of next-generation sequencing using the ABI SOLiD technology to mammalian transcriptome analysis enabled a survey of the content, the complexity and the developmental dynamics of the embryonic stem cell transcriptome in the mouse. Also in this issue, Mortazavi et al. report Illumina technology–based RNA-Seq analysis of the mouse transcriptome in three different tissues.
Mapping and quantifying mammalian transcriptomes by RNA-Seqpp621 - 628 Ali Mortazavi, Brian A Williams, Kenneth McCue, Lorian Schaeffer & Barbara Wold Published online: 30 May 2008|doi:10.1038/nmeth.1226
The mouse transcriptome in three tissue types has been analyzed using Illumina next-generation sequencing technology. This quantitative RNA-Seq methodology has been used for expression analysis and splice isoform discovery and to confirm or extend reference gene models. Also in this issue, another paper reports application of the ABI SOLiD technology to sequence the transcriptome in mouse embryonic stem cells.
Efficient targeted transcript discovery via array-based normalization of RACE librariespp629 - 635 Sarah Djebali, Philipp Kapranov, Sylvain Foissac, Julien Lagarde, Alexandre Reymond, Catherine Ucla, Carine Wyss, Jorg Drenkow, Erica Dumais, Ryan R Murray, Chenwei Lin, David Szeto, France Denoeud, Miquel Calvo, Adam Frankish, Jennifer Harrow, Periklis Makrythanasis, Marc Vidal, Kourosh Salehi-Ashtiani, Stylianos E Antonarakis, Thomas R Gingeras & Roderic Guigó Published online: 25 May 2008|doi:10.1038/nmeth.1216 Abstract|Full text|PDF
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|Supplementary Information See also:News and Views by Carninci
Automated on-chip rapid microscopy, phenotyping and sorting of C. eleganspp637 - 643 Kwanghun Chung, Matthew M Crane & Hang Lu Published online: 22 June 2008|doi:10.1038/nmeth.1227
A major bottleneck for genetic approaches in model organisms is the application of state-of-the-art technologies to phenotyping. Now, using a microfluidic chip, high-resolution imaging of fluorescent reporters and accurate sorting is demonstrated in an automated manner in Caenorhabditis elegans.
Functional immobilization of signaling proteins enables control of stem cell fatepp645 - 650 Kristin Alberti, Ryan E Davey, Kento Onishi, Sophia George, Katrin Salchert, F Philipp Seib, Martin Bornhäuser, Tilo Pompe, Andras Nagy, Carsten Werner & Peter W Zandstra Published online: 15 June 2008|doi:10.1038/nmeth.1222
Cells in vivo are exposed not only to soluble factors but also to immobilized ligands. Controlled immobilization of functional growth factors yields dose-dependent responses in mouse embryonic stem cells in vitro and allows the effects of immobilized versus soluble ligands to be studied.