Measurement of low-abundance cytokine mRNA in cells of murine lymphoid organs: a new quantitative reverse transcription/polymerase chain reaction method.
To investigate cytokine regulation in cells of freshly excised lymphoid tissues, rigorous quantitative reverse transcription/polymerase chain reaction (QRT-PCR) assays were developed to measure attomole (10(-18) mol) amounts of the mRNA for seven cytokines: interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), transforming growth factor beta (TGFbeta), IL-2 and IL-6. RNA was purified from single-cell suspensions of immune tissues (spleen, thymus and resident peritoneal cells). Data are presented demonstrating the utility of these assays for quantifying basal levels of all seven cytokine mRNAs in the freshly isolated splenocytes and thymocytes. Studies to establish the usefulness of these assays for measuring changes in the levels of cytokine mRNA focused on IL-1alpha, IL-1beta, TNFalpha and IL-2 in splenocytes, thymocytes and resident peritoneal cells. Using the QRT-PCR assays developed, levels of cytokine mRNA could be quantified in RNA samples obtained both from freshly isolated cells and from cells following short-term (</=26 h) culture. These measurements established basal in vivo levels of specific cytokine mRNAs and demonstrated specific modulation of their levels by cell culture and by the inclusion of immune stimulants (bacterial lipopolysaccharide or the plant lectin concanavalin A) in the culture. These data provide new information on both basal and stimulated cytokine levels that is required for valid interpretations of the roles of cytokine expression in immune regulation.
Berleth
E S
ES
Department of Pharmacology, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Ujhazy
P
P
Meer
J M
JM
Dolnick
B J
BJ
Ehrke
M J
MJ
D000818:Animals; D001202:Ascitic Fluid / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis* / Q000235:genetics; D005260:Female; D007375:Interleukin-1 / Q000378:metabolism; D008221:Lymphoid Tissue / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D011786:Quality Control; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016207:Cytokines; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha