Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction.

TLDR

GM‑CSF and IL‑3 expression in MLA‑144 cells was examined before and after induction with phorbol 12‑myristate 13‑acetate. The study adapts PCR for precise quantitation of mRNA or DNA from a small number of cells. The method uses competitive PCR with a competitor DNA differing by an intron or restriction site, amplifying both target and competitor with the same primers to keep product ratios constant for accurate quantitation. The technique yields constant product ratios, detects GM‑CSF and IL‑3 mRNA only after induction in as few as 200 cells, and accurately measures GM‑CSF gene copy number in normal, 5q‑, and hybrid cell lines.

Abstract

The expression of two cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3), has been investigated in MLA-144 cells before and after induction with phorbol 12-myristate 13-acetate. We describe an adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells. Aliquots of the PCR mixture containing cDNA copies of the RNA to be assayed were added to serial dilutions of a competitor DNA fragment that differed from the cDNA of interest by having either a small intron or a mutated internal restriction enzyme site. Therefore, the same primers were used to coamplify the unknown and the competitor. The ratio of products remains constant through the amplification and can be readily quantitated. In unstimulated cells, no GM-CSF or IL-3 mRNA could be detected. However, with appropriate induction, mRNA for both cytokines was detected and quantitated in as few as 200 cells. Competitive PCR was also used to accurately quantitate the copy number of the human GM-CSF gene in normal human cells, in a clonal population of cells from a patient with 5q- syndrome, and in a human-hamster cell line known to have only one copy of the human GM-CSF gene.