A sensitive, non-radioactive quantitative method for measuring IL-4 and IL-4delta2 mRNA in unstimulated cells from multiple clinical samples, using nested RT-PCR.
Measurements of IL-4 mRNA directly from clinical samples are technically difficult as IL-4 is a low copy number cytokine. Moreover, most existing studies involving RT-PCR are confused by the use of primers which simultaneously amplify cDNA of IL-4 and its splice-variant (IL-4delta2). We describe a sensitive nested RT-PCR method to quantify mRNA expression of IL-4 and IL-4delta2 separately. It involves a simple method of generating cRNA standards without cloning. The use of external synthetic RNA standards, for which we validate that amplification kinetics are equivalent to the target, obviates the need for multiple sample dilutions. The assay is sensitive enough to measure IL-4 and IL-4delta2 mRNA expression in unstimulated PBMCs of normal subjects, and the reproducibility and throughput make this assay suitable for use in clinical studies with multiple samples.
Seah
G T
GT
Department of Bacteriology, Windeyer Institute of Medical Sciences, Royal Free and University College School of Medicine, Windeyer Building, 46 Cleveland Street, London, UK.
Rook
G A
GA
D017398:Alternative Splicing; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D006801:Humans; D015847:Interleukin-4 / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000276:immunology; D018075:RNA, Complementary / Q000235:genetics / Q000592:standards; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018075:RNA, Complementary; D012333:RNA, Messenger; D015847:Interleukin-4