A method for rapid analysis of peripheral blood mononuclear leukocyte cytokine mRNA.
Interleukins, hematopoietic growth factors, and adhesion molecules mediate multiple components of the inflammatory response. Conventional methods for purification of cytokine-producing cells are time consuming and can, therefore, depending on the conditions of cell purification and the nature of the mRNA being purified, result in both over- and underestimates of mRNA content. Therefore, it has been difficult to quantify such factors in ways that are unambiguously reflective of in vivo cytokine production. We describe herein a method for such rapid preparation of peripheral blood mononuclear leukocytes (PBML) that amounts of cytokine mRNA derived from these cells, apart from readily quantifiable losses during RNA preparation, will be reflective of quantities in vivo. Small quantities (10(5) of normal PBML were isolated by density centrifugation of heparinized blood for 1.25 min in a capillary tube. Poly(A)+ RNA isolated by oligo(dT)-cellulose column chromatography was sufficient to reverse transcribe both antigen-specific T-cell-receptor beta-chain mRNA and interleukin 6 (IL-6) mRNA and subsequently amplify the cDNA with Taq polymerase in reverse transcription-polymerase chain reactions (RT-PCR). Although IL-6 mRNA was not present in normal PBML, PBML incubated in culture medium for only 3 h contained approximately one molecule per cell. Because of its rapidity this technique will permit quantification of cytokine mRNA in the steady state and in clinical setting of inflammation. Because the method requires only a small quantity of blood it can be applied to clinical research studies involving children.
Bickler
S W
SW
Department of Surgery, Oregon Health Sciences University, Portland.
Heinrich
M C
MC
Davey
M
M
Harrison
M W
MW
Bagby
G C
GC
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D016207:Cytokines / Q000235:genetics*; D004586:Electrophoresis; D005784:Gene Amplification; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics
D016207:Cytokines; D009841:Oligonucleotides; D012333:RNA, Messenger