Global cDNA amplification combined with real-time RT-PCR: Accurate quantification of multiple human potassium channel genes at the single cell levelCitation formats

  • Authors:
  • A. Ai-Taher
  • A. Bashein
  • T. Nolan
  • M. Hollingsworth
  • G. Brady

Standard

Global cDNA amplification combined with real-time RT-PCR: Accurate quantification of multiple human potassium channel genes at the single cell level. / Ai-Taher, A.; Bashein, A.; Nolan, T.; Hollingsworth, M.; Brady, G.

In: Yeast, Vol. 17, No. 3, 2000, p. 201-210.

Research output: Contribution to journalArticlepeer-review

Harvard

APA

Vancouver

Author

Ai-Taher, A. ; Bashein, A. ; Nolan, T. ; Hollingsworth, M. ; Brady, G. / Global cDNA amplification combined with real-time RT-PCR: Accurate quantification of multiple human potassium channel genes at the single cell level. In: Yeast. 2000 ; Vol. 17, No. 3. pp. 201-210.

Bibtex

@article{f7f3d89e94244ae4855c825bc9d96c34,
title = "Global cDNA amplification combined with real-time RT-PCR: Accurate quantification of multiple human potassium channel genes at the single cell level",
abstract = "We have developed a sensitive quantitative RT-PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel niRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT-PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use of TaqMan{\texttrademark} real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than l/100th copy/cell (one specific cDNA molecule present amongst 10 8 total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis. Copyright {\textcopyright}2000 John Wiley & Sons, Ltd.",
keywords = "Quantitative, Real-time PCR, RT-PCR, Single-cell",
author = "A. Ai-Taher and A. Bashein and T. Nolan and M. Hollingsworth and G. Brady",
year = "2000",
doi = "10.1002/1097-0061(20000930)17:3<201::AID-YEA30>3.0.CO;2-R",
language = "English",
volume = "17",
pages = "201--210",
journal = "Yeast",
issn = "0749-503X",
publisher = "John Wiley & Sons Ltd",
number = "3",

}

RIS

TY - JOUR

T1 - Global cDNA amplification combined with real-time RT-PCR: Accurate quantification of multiple human potassium channel genes at the single cell level

AU - Ai-Taher, A.

AU - Bashein, A.

AU - Nolan, T.

AU - Hollingsworth, M.

AU - Brady, G.

PY - 2000

Y1 - 2000

N2 - We have developed a sensitive quantitative RT-PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel niRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT-PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use of TaqMan™ real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than l/100th copy/cell (one specific cDNA molecule present amongst 10 8 total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis. Copyright ©2000 John Wiley & Sons, Ltd.

AB - We have developed a sensitive quantitative RT-PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel niRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT-PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use of TaqMan™ real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than l/100th copy/cell (one specific cDNA molecule present amongst 10 8 total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis. Copyright ©2000 John Wiley & Sons, Ltd.

KW - Quantitative

KW - Real-time PCR

KW - RT-PCR

KW - Single-cell

U2 - 10.1002/1097-0061(20000930)17:3<201::AID-YEA30>3.0.CO;2-R

DO - 10.1002/1097-0061(20000930)17:3<201::AID-YEA30>3.0.CO;2-R

M3 - Article

C2 - 0011025530

VL - 17

SP - 201

EP - 210

JO - Yeast

JF - Yeast

SN - 0749-503X

IS - 3

ER -