Use of quantitative polymerase chain reaction to quantitate cytokine messenger RNA molecules.
A quantitative polymerase chain reaction (PCR) using an internal control (Standard) RNA was developed for precise quantitation of human cytokine mRNA. The target mRNA and internal control were simultaneously reverse transcribed and co-amplified using the same set of primers. The amount of specific target mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The internal control RNA consisted of linearly connected sequences of the 5' primers of multiple cytokine genes followed by the complementary sequences to their 3' primers in the same order. This structure of the internal standard enables one to use the same internal standard for quantitating multiple cytokine mRNAs. Using this approach, we estimated the induced levels of cytokine mRNA, IL2, IL6, and TNF-alpha to be 2.8 x 10(6), 2.4 x 10(5) and 3.4 x 10(8) molecules per 1.0 microgram total cellular RNA of Jurkat, THP-1 and HL 60 cells, respectively. Comparable values were obtained when quantitation experiments were done on another batch of THP-1 and HL-60 total cellular RNA. The excellent sensitivity and reproducibility makes this approach a valuable one in following changes in cytokine gene expression in wide variety of conditions, both in vivo and in vitro.
Kanangat
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Department of Microbiology, College of Veterinary Medicine, University of Tennessee, Knoxville 37996-0845.
Solomon
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A
Rouse
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BT
D000199:Actins / Q000096:biosynthesis; D002460:Cell Line; D016207:Cytokines / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D008070:Lipopolysaccharides; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013755:Tetradecanoylphorbol Acetate; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D000199:Actins; D016207:Cytokines; D007376:Interleukin-2; D015850:Interleukin-6; D008070:Lipopolysaccharides; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D013755:Tetradecanoylphorbol Acetate