TY - JOUR
T1 - A synthetic standard DNA construct for use in quantification of murine cytokine mRNA molecules
AU - David Farrar, J.
AU - Street, Nancy E.
N1 - Funding Information:
Acknowledgements-The authors thank Dr Gerald Thrush, KathleenH oag,J ana Windsor and Kathy Katz for constructive scientificd iscussionsW. e areg ratefult o Dr Donald Capra and Shirley Hall for DNA sequencingo f the initial recombinants. We greatly appreciateD r Richard Scheuermannf or expert advice in designing the construct and for many helpful discussionsW. e thank Dr Jerry Y. Niederkorna nd Dr Richard Scheuermannfo r criticallyr eviewingt hem anuscriptT. his work wass upportedin part by a grantf rom theN ational Instituteso f Health NC1 5 R29 CA55266-04a nd a grant from the National CancerI nstituteN C1 ST32 CA09082.
PY - 1995/9
Y1 - 1995/9
N2 - A synthetic DNA construct has been developed as a standard molecule whereby murine cytokine mRNA molecules can be quantified by the reverse transcription-polymerase chain reaction (RT-PCR). The construct, designated Cytoquant 1, allows the quantification of murine IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-γ, TNF-α, TGF-β, GM-CSF, CD4, CD8, HPRT and β-actin mRNA levels. This technique is based on the amplification of a transcribed RNA molecule from Cytoquant 1 as an internal standard control in both the RT and PCR reactions. The quantification data from these analyses are expressed in absolute values, i.e. molecules/cell, which allows the data derived from separate experiments to be compared. In this study, mRNAs encoding β-actin, IL-10, IFN-γ and GM-CSF have been quantitated in both Th1 and Th2 cell clones with, and without, stimulation. The quantitative analysis data are highly reproducible and cytokine mRNA concentrations are reflective of restricted cytokine secretion patterns. Furthermore, constitutive cytokine mRNA levels are detectable in resting cells, eliminating the need for exogenous stimulation. The high degree of sensitivity and accuracy make this methodology uniquely suited for the study of T-cell subset cytokine expression in both in vivo and in vitro biological models.
AB - A synthetic DNA construct has been developed as a standard molecule whereby murine cytokine mRNA molecules can be quantified by the reverse transcription-polymerase chain reaction (RT-PCR). The construct, designated Cytoquant 1, allows the quantification of murine IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-γ, TNF-α, TGF-β, GM-CSF, CD4, CD8, HPRT and β-actin mRNA levels. This technique is based on the amplification of a transcribed RNA molecule from Cytoquant 1 as an internal standard control in both the RT and PCR reactions. The quantification data from these analyses are expressed in absolute values, i.e. molecules/cell, which allows the data derived from separate experiments to be compared. In this study, mRNAs encoding β-actin, IL-10, IFN-γ and GM-CSF have been quantitated in both Th1 and Th2 cell clones with, and without, stimulation. The quantitative analysis data are highly reproducible and cytokine mRNA concentrations are reflective of restricted cytokine secretion patterns. Furthermore, constitutive cytokine mRNA levels are detectable in resting cells, eliminating the need for exogenous stimulation. The high degree of sensitivity and accuracy make this methodology uniquely suited for the study of T-cell subset cytokine expression in both in vivo and in vitro biological models.
KW - T-helper subset
KW - cytokine
KW - quantitative PCR
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U2 - 10.1016/0161-5890(95)00061-I
DO - 10.1016/0161-5890(95)00061-I
M3 - Article
C2 - 7477005
AN - SCOPUS:0028874523
SN - 0161-5890
VL - 32
SP - 991
EP - 1000
JO - Molecular Immunology
JF - Molecular Immunology
IS - 13
ER -