Quantification of murine IFN-gamma mRNA and protein expression: impact of real-time kinetic RT-PCR using SYBR green I dye.
Reliable quantification of cytokine mRNA expression is an important technique for analyzing immune responses. Up until now, little to no information has been available as to whether different mRNA quantification techniques lead to similar results. Recently, real time quantitative reverse transcriptase (RT)-PCR using SYBR Green I as a double stranded DNA specific dye has been introduced. This novel method enables simple and rapid measurement of PCR product accumulation during the log-linear reaction phase and obviates the need for expensive hybridization probes. Here, we analyzed murine gamma interferon (IFN)-gamma mRNA expression in splenocytes by this technique in comparison to semiquantitative noncompetitive RT-PCR, Northern blot analysis, and ELISA after stimulation of the cells with interleukin (IL)-12, IL-18 and a combination of both cytokines. The results clearly show that all of the techniques detect differences in the IFN-gamma gene expression induced by these distinctive stimuli qualitatively exactly in the same order. However, real-time kinetic RT-PCR offers several advantages, notably its high sensitivity that allows the detection of basal IFN-gamma mRNA expression in unstimulated samples. In addition it provides the lowest interassay variability of all techniques investigated. Finally, the gene expression measured by this method eliminates any post-PCR manipulations because the PCR product identification can be easily performed by melting curve analysis.
Hein
J
J
Institut für Klinische Immunologie und Transfusionsmedizin, Justus-von-Liebig-Universität, Langhansstrasse 7, D-35385 Giessen, Germany. joachim.hein@mikrobio.med.uni-erlangen.de
Schellenberg
U
U
Bein
G
G
Hackstein
H
H
D000818:Animals; D052160:Benzothiazoles; D015152:Blotting, Northern; D002478:Cells, Cultured; D003959:Diamines; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry*; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I; D007371:Interferon-gamma