Semi-quantification of human C-C chemokine mRNAs with reverse transcription/real-time PCR using multi-specific standards.
J Immunol Methods
; 241(1-2): 109-19, 2000 Jul 31.
Article
em En
| MEDLINE
| ID: mdl-10915853
ABSTRACT
A reverse transcription/real-time polymerase chain reaction (PCR) assay was established to semi-quantify the mRNA levels of the human C-C chemokines RANTES, MIP-1beta and MCP-1 relative to the housekeeping gene beta-actin. The assay showed a high sensitivity (below 60 cDNA molecules/10 microl reaction) and dynamic range (8 log units); both within-assay and inter-assay variability were below 0.06 log units and the accuracy was +/-0.06 log units for all four chemokines. Moreover, it is demonstrated that a multi-specific DNA fragment, which had previously been constructed for competitive PCR, can be used as a reliable external standard. This allows a direct semi-quantitative comparison of different chemokine mRNA levels and is a convenient alternative to the use of different sets of homologous external standards. The method was successfully applied to the semi-quantification of chemokines in human liver specimens and should be useful in further studies on steady state mRNA levels of C-C chemokines from low cell numbers or small tissue specimens.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
/
Quimiocinas CC
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Limite:
Humans
Idioma:
En
Ano de publicação:
2000
Tipo de documento:
Article