Rapid and sensitive mRNA phenotyping for interleukins (IL-1 to IL-6) and colony-stimulating factors (G-CSF, M-CSF, and GM-CSF) by reverse transcription and subsequent polymerase chain reaction.
Oligonucleotide primer pairs specific for interleukins (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, and IL-6, as well as for granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA/cDNA were synthesized in order to detect cytokine transcripts by reverse transcription and subsequent polymerase chain reaction (RT/PCR). Analysis of RNA preparations of the human bladder carcinoma cell line 5637 by this methodology reveals expression of mRNAs for IL-1 alpha, IL-1 beta, IL-6, G-CSF, M-CSF, and for GM-CSF, whereas mRNAs for IL-2, IL-3, IL-4, and IL-5 are not detectable. These results are in agreement with data obtained by classical methods. Thus, for the cytokines IL-2, IL-3, IL-4, and IL-5, it was not possible to detect a phenomenon described as 'illegitimate transcription,' defined as the low level transcription of any gene in any cell type. This finding is of importance for the applicability of mRNA phenotyping employing RT/PCR for the determination of mRNA expression patterns. For M-CSF mRNA detection, two oligonucleotide primer pairs had to be used to distinguish between the alpha-(pcCSF17) and beta-splicing forms and to overcome the problem of non-amplification of a larger fragment in the presence of a competing smaller one, defined here as 'incomplete positivity.' For G-CSF, IL-4, IL-2, and IL-5, RT/PCR reveals two fragments. Restriction enzyme analysis of the additional fragments suggests that they may arise from alternative splicing events. For G-CSF and IL-4, exons 3 and 2 seem to be spliced out, respectively. The additional fragments for IL-2 and IL-5 RT/PCR have not yet been further characterized, but the size of the fragments makes it seem probable that exons 2 and 3 are spliced out for IL-2 and IL-5, respectively. The biological role of these alternative mRNAs has yet to be determined.
Sorg
R
R
Immunology Laboratory, Heinrich-Heine-University, Düsseldorf, Germany.
Enczmann
J
J
Sorg
U
U
Heermeier
K
K
Schneider
E M
EM
Wernet
P
P
D001483:Base Sequence; D003115:Colony-Stimulating Factors / Q000235:genetics*; D006801:Humans; D007378:Interleukins / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000378:metabolism
D003115:Colony-Stimulating Factors; D007378:Interleukins; D012333:RNA, Messenger