PMID- 17657627
OWN - NLM
STAT- PubMed-not-MEDLINE
DCOM- 20100112
LR  - 20181113
IS  - 0925-4692 (Print)
IS  - 0925-4692 (Linking)
VI  - 6
IP  - 4
DP  - 1998
TI  - Semiquantitative determination of human cytokine mRna expression using TaqMan 
      RT-PCR.
PG  - 297-309
AB  - To establish an easy, fast and reliable RT-PCR for the analysis of human cytokine 
      expression, we made use of the recently developed technique of TaqMan PCR. This 
      technique is based on the cleavage of fluorochrome-labelled internal 
      oligodeoxynucleotide probes by the 5'-->3' nuclease activity of Taq DNA 
      polymerase. Measurement of fluorescence intensity during each cycle of the PCR 
      reaction with a Sequence Detection System allows the determination of a threshold 
      cycle at which an increase in fluorescence intensity is first detectable. From 
      these values, a starting amount of template DNA can be calculated. Here, we 
      established specific primers and corresponding internal, fluorogenic probes for 
      the human cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta 
      (IL-1beta) and interferon-gamma (IFN-gamma), and for the constant region of the 
      T-cell receptor beta chain (TCRbeta) and the housekeeping gene, 
      glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) for normalization of mRNA 
      expression levels. Titrations of the cDNA input showed a strict inverse 
      correlation between the threshold cycles obtained and the starting amount of 
      template. This in turn allowed the generation of a standard curve, and thus 
      quantification of mRNA abundance in cDNA samples. Evaluation of the method using 
      cDNAs from peripheral blood mononuclear cells (PBMC) stimulated with 
      lipopolysaccharide (LPS) or phytohae-magglutinin (PHA) showed basal expression of 
      TNF-alpha and IL-1beta in untreated PBMC while IFN-y was not detectable or only 
      weakly expressed. After stimulation with LPS, a strong induction of IL-1beta and 
      TNF-alpha was measured, while IFN-gamma was induced to a lesser extent. PHA 
      treatment, in contrast, led to an induction of all three cytokines with IFN-gamma 
      being the most prominent. The method has a large dynamic range, requires no 
      post-PCR processing and gives reliable results.
FAU - Lang, R
AU  - Lang R
AD  - Institute for Medical Microbiology, Immunology and Hygiene, Technical University 
      of Munich, Munich, Germany.
FAU - Heeg, K
AU  - Heeg K
LA  - eng
PT  - Journal Article
PL  - Switzerland
TA  - Inflammopharmacology
JT  - Inflammopharmacology
JID - 9112626
EDAT- 2007/07/28 09:00
MHDA- 2007/07/28 09:01
CRDT- 2007/07/28 09:00
PHST- 1998/08/07 00:00 [received]
PHST- 1998/09/23 00:00 [accepted]
PHST- 1998/08/17 00:00 [revised]
PHST- 2007/07/28 09:00 [pubmed]
PHST- 2007/07/28 09:01 [medline]
PHST- 2007/07/28 09:00 [entrez]
AID - 10.1007/s10787-998-0014-4 [doi]
PST - ppublish
SO  - Inflammopharmacology. 1998;6(4):297-309. doi: 10.1007/s10787-998-0014-4.