Quantification of IL-2 and IL-2 receptor mRNA in peripheral blood mononuclear cells by the RT-PCR based SHARP Signal system.
Nuclear transcription assays have shown that increases in interleukin 2 (IL-2) and its receptor (IL-2R) mRNA are reflected at the level of transcription. However, the quantification of transient and low-level expression of IL-2/IL-2R mRNAs in normal resting peripheral blood mononuclear cells (PBMC) requires a sensitive and reliable assay. We have established a quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) assay to measure IL-2/IL-2R transcripts by modifying the commercially-available SHARP Signal system to include IL-2/IL-2R RNA probes that were constructed by in vitro transcription of phagemid clones. To evaluate this modified SHARP Signal system and to demonstrate its clinical utility, the expression levels of IL-2 and IL-2R were assessed for 40 healthy normal donors. The mean +/- SEM levels of transcripts in normal PBMC expressed in zeptomol per micrograms total RNA for IL-2, IL-2R alpha and IL-2R beta were 2.6 +/- 0.5, 23.3 +/- 2.2 and 157.2 +/- 32.2 respectively. Compared with the conventional RT-PCR and gel-electrophoresis-based detection method, the SHARP Signal system is fast, not labor-intensive and inexpensive, and can be readily adapted for the measurement of other cytokines or cytokine receptor gene expressions in a clinical diagnostic laboratory environment without extensive experience in molecular techniques.
Lee
B N
BN
Section of Transfusion Medicine and Laboratory Immunology, University of Texas, MD Anderson Cancer Center, Houston 77030, USA.
Lu
J G
JG
Shen
D Y
DY
Li
S
S
Shearer
W T
WT
Reuben
J M
JM
D000328:Adult; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000592:standards; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015347:RNA Probes; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics*; D015375:Receptors, Interleukin-2 / Q000235:genetics*; D012015:Reference Standards; D012016:Reference Values
D017931:DNA Primers; D018076:DNA, Complementary; D007376:Interleukin-2; D015347:RNA Probes; D012333:RNA, Messenger; D015375:Receptors, Interleukin-2