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John L. Dart Library
9 a.m. – 7 p.m.
Phone: (843) 722-7550
West Ashley Library
9 a.m. – 7 p.m.
Phone: (843) 766-6635
Folly Beach Library
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Phone: (843) 588-2001
Edgar Allan Poe/Sullivan's Island Library
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Phone: (843) 883-3914
Wando Mount Pleasant Library
9 a.m. – 8 p.m.
Phone: (843) 805-6888
Village Library
9 a.m. – 6 p.m.
Phone: (843) 884-9741
St. Paul's/Hollywood Library
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McClellanville Library
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Comparison of reverse transcription–quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis
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- Author(s): Fox, Bridget C.1; Devonshire, Alison S. ; Baradez, Marc-Olivier1; Marshall, Damian1; Foy, Carole A.1
- Source:
Analytical Biochemistry. Aug2012, Vol. 427 Issue 2, p178-186. 9p.- Subject Terms:
- Source:
- Additional Information
- Abstract: Abstract: Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) is the “gold standard” for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT–qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. [Copyright &y& Elsevier]
- Abstract: Copyright of Analytical Biochemistry is the property of Academic Press Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
- Abstract:
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