Citation

Novel Protein Expression Assays for the Detection and Relative Quantification of Proteins in Human Embryonic Stem Cells using qPCR

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Abstract:

Carol Khodier,Tina Settineri, Elana Swartzman, Mark Shannon, Shiaw-Min Chen and David Ruff
Applied Biosystems, part of Life Technologies Corporation

Current methods for detection of protein markers in stem cells include FACS, immunohistochemistry and western blots. Each method, however, has its own limitations such as requirement for large sample input, lack of sensitivity, or is not inherently quantitative. Proximity ligation assay (PLA) technology extends qPCR applications now to the detection of cellular proteins through the amplification of a surrogate DNA template. PLA is a three-step process that involves, 1) binding of paired antibody-oligonucleotide probes to a protein target in biological samples, 2) templated ligation of the oligonucleotides in proximity, and 3) qPCR detection. We have optimized this technique for crude cell lysates utilizing a simple, one-step sample lysis approach to release all classes of proteins, and combined it with gold standard TaqMan® chemistry to create a highly sensitive and specific process for measuring protein expression in small samples. One application of this assay is the detection and relative quantification of markers in pluripotent and differentiated stem cells. Stem cell characterization typically relies on determining the presence and amount of stage specific protein markers such as OCT4, NANOG, SOX2, and LIN28. Protein expression results confirm cell-stage specific changes in protein expression of these key stem cell markers, and the data can be directly compared with published mRNA expression profiles for the same cell lines. We have engaged a number of researchers as test sites for these assays and are gathering input and feedback. Our findings illustrate how this new assay system expands the scope of qPCR to protein detection and quantification, an important area of cell biology.
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Association:
Name: Connecticut's Stem Cell Research International Symposium
URL:
http://stemconn.org


Citation:
URL: http://citation.allacademic.com/meta/p319952_index.html
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MLA Citation:

Khodier, Carol. "Novel Protein Expression Assays for the Detection and Relative Quantification of Proteins in Human Embryonic Stem Cells using qPCR" Paper presented at the annual meeting of the Connecticut's Stem Cell Research International Symposium, Omni Hotel, New Haven, CT, Mar 23, 2009 <Not Available>. 2014-11-29 <http://citation.allacademic.com/meta/p319952_index.html>

APA Citation:

Khodier, C. , 2009-03-23 "Novel Protein Expression Assays for the Detection and Relative Quantification of Proteins in Human Embryonic Stem Cells using qPCR" Paper presented at the annual meeting of the Connecticut's Stem Cell Research International Symposium, Omni Hotel, New Haven, CT <Not Available>. 2014-11-29 from http://citation.allacademic.com/meta/p319952_index.html

Publication Type: Poster
Review Method: Peer Reviewed
Abstract: Carol Khodier,Tina Settineri, Elana Swartzman, Mark Shannon, Shiaw-Min Chen and David Ruff
Applied Biosystems, part of Life Technologies Corporation

Current methods for detection of protein markers in stem cells include FACS, immunohistochemistry and western blots. Each method, however, has its own limitations such as requirement for large sample input, lack of sensitivity, or is not inherently quantitative. Proximity ligation assay (PLA) technology extends qPCR applications now to the detection of cellular proteins through the amplification of a surrogate DNA template. PLA is a three-step process that involves, 1) binding of paired antibody-oligonucleotide probes to a protein target in biological samples, 2) templated ligation of the oligonucleotides in proximity, and 3) qPCR detection. We have optimized this technique for crude cell lysates utilizing a simple, one-step sample lysis approach to release all classes of proteins, and combined it with gold standard TaqMan® chemistry to create a highly sensitive and specific process for measuring protein expression in small samples. One application of this assay is the detection and relative quantification of markers in pluripotent and differentiated stem cells. Stem cell characterization typically relies on determining the presence and amount of stage specific protein markers such as OCT4, NANOG, SOX2, and LIN28. Protein expression results confirm cell-stage specific changes in protein expression of these key stem cell markers, and the data can be directly compared with published mRNA expression profiles for the same cell lines. We have engaged a number of researchers as test sites for these assays and are gathering input and feedback. Our findings illustrate how this new assay system expands the scope of qPCR to protein detection and quantification, an important area of cell biology.


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