A competitive RT-PCR method for the quantitative analysis of cytokine mRNAs in mouse tissues.
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.
Zhou
N M
NM
Rega Institute for Medical Research, University of Leuven, Belgium.
Matthys
P
P
Polacek
C
C
Fiten
P
P
Sato
A
A
Billiau
A
A
Froyen
G
G
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology; D016207:Cytokines / Q000032:analysis* / Q000235:genetics; D007371:Interferon-gamma / Q000235:genetics; D016753:Interleukin-10 / Q000235:genetics; D015847:Interleukin-4 / Q000235:genetics; D007834:Lasers; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012016:Reference Values; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013154:Spleen / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D000906:Antibodies; D017252:CD3 Complex; D016207:Cytokines; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D015847:Interleukin-4; D007371:Interferon-gamma