A competitive RT-PCR method for the quantitative analysis of cytokine mRNAs in mouse tissues.

Abstract:

:The authors describe the design and validation of a competitive RT-PCR method for the efficient and reproducible quantitation of mRNA molecules of IFN-gamma, TNF-alpha, IL-4 and IL-10 in mouse spleen RNA extracts. Before being subjected to RT-PCR, the RNA extracts were supplemented with internal control RNAs (IC-RNAs), which were constructed by inserting DNA fragments in the cDNA of the respective cytokines. The efficiency of amplification of the target and the IC-RNA was shown to remain equal over a wide range of cycle numbers. Reproducibility was such that differences in mRNA contents that were greater than 17% could be detected between two RNA samples run in parallel. Normal mouse spleen tissue was found to contain 10(7)-10(8) molecules of TNF-alpha, IFN-gamma, IL-4 and IL-10 mRNA per micrograms total RNA extracted. Injection of animals with anti-CD3 antibody, a well-known cytokine inducer, resulted in a moderate increase in TNF-alpha and IL-10 mRNA levels (14- and 24-fold, respectively), and in a substantially greater increase in the levels of mRNA for IL-4 and IFN-gamma (199- and 851-fold, respectively). These results demonstrate an accurate and reliable quantitation of cytokine mRNA levels in animal tissues.

journal_name

Cytokine

journal_title

Cytokine

authors

Zhou NM,Matthys P,Polacek C,Fiten P,Sato A,Billiau A,Froyen G

doi

10.1006/cyto.1996.0156

subject

Has Abstract

pub_date

1997-03-01 00:00:00

pages

212-8

issue

3

eissn

1043-4666

issn

1096-0023

pii

S1043-4666(96)90156-8

journal_volume

9

pub_type

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