A simplified, competitive RT-PCR method for measuring rat IFN-gamma mRNA expression.
We describe an adaptation of competitive RT-PCR to quantitate rat IFN-gamma mRNA expression. An IFN-gamma DNA mimic that shared the same primers and had an identical sequence to the target mRNA except for deletion of 66 nucleotides was created by a simple PCR amplification from target cDNA. To reduce variations of initial RNA concentrations, beta-actin cDNAs from each target RNA sample were normalized using the densitometric data. A known amount of pretitrated DNA competitor was then used to analyze the relative levels of target cDNA in different samples by PCR co-amplification. The amplification efficiency for both target and competitor remained constant throughout the PCR reaction, and the ratio of target to competitor PCR product remained proportional to the initial ratio of target to competitor. Relative mRNA levels among samples determined by this method were comparable to levels determined by northern blot analysis. They were also comparable to levels of IFN-gamma protein estimated by ELISA. We conclude that this method can be used to estimate the relative abundance of the target mRNA. This method is adaptable to quantitation of other cytokines and is particularly valuable if there are numerous samples or if the amount of initial mRNA is limited.
Sun
B
B
Section of Immunoregulation, National Eye Institute, NIH, Bethesda, MD 20892, USA.
Wells
J
J
Goldmuntz
E
E
Silver
P
P
Remmers
E F
EF
Wilder
R L
RL
Caspi
R R
RR
D000818:Animals; D007371:Interferon-gamma / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D051381:Rats; D011916:Rats, Inbred F344; D013601:T-Lymphocytes / Q000378:metabolism
D012333:RNA, Messenger; D007371:Interferon-gamma