Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR.

Abstract:

:Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.

journal_name

Cytokine

journal_title

Cytokine

authors

Overbergh L,Valckx D,Waer M,Mathieu C

doi

10.1006/cyto.1998.0426

keywords:

subject

Has Abstract

pub_date

1999-04-01 00:00:00

pages

305-12

issue

4

eissn

1043-4666

issn

1096-0023

pii

S1043-4666(98)90426-4

journal_volume

11

pub_type

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