Measurement of feline cytokine gene expression by quantitative-competitive RT-PCR.
We have developed a method to quantitate feline cytokine gene expression using competitive RT-PCR. Feline cytokine specific primers were developed that encompass an intron, thus allowing differentiation of cDNA vs. genomic DNA amplification products. The PCR products of the primers were verified by sequencing and Southern blot analysis. For quantitation, a non-homologous RNA competitor was created for each cytokine of interest. The competitor was designed to yield an RT-PCR product 10-20% larger than the native sequence, thereby allowing differentiation of the two products by electrophoresis on an agarose gel. Both competitor and native sequences used the same primer sequences for RT (oligo dT) and PCR (cytokine specific). The amplification efficiency of the competitor and native sequence was shown to be identical which allowed comparison at any point during the amplification, including the plateau phase. The quantity of starting cytokine mRNA was determined by interpolation from a standard curve. As little as 1 microgram of total cellular RNA was required per cytokine determination. The assay can routinely quantify as few as 1000 copies of template and spans a range of up to 4 log.
Dean
G A
GA
Department of Microbiology, Pathology, and Parasitology, North Carolina State University, Raleigh 27606, USA. gregg_dean@ncsu.edu
Higgins
J
J
LaVoy
A
A
Fan
Z
Z
Pedersen
N C
NC
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002415:Cats / Q000235:genetics* / Q000276:immunology*; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000662:veterinary*; D012333:RNA, Messenger / Q000235:genetics; D012015:Reference Standards
D016207:Cytokines; D017931:DNA Primers; D012333:RNA, Messenger