Rapid nonradioactive in situ hybridization for interleukin-2 mRNA with riboprobes generated using the polymerase chain reaction.
In situ hybridization is a technique with widespread application. However, its usefulness has been limited by the need for radioactive materials and the requirement for the DNA to be cloned onto an appropriate vector. We have utilized the polymerase chain reaction to directly incorporate a T7 RNA polymerase promoter sequence onto the cDNA for interleukin-2. Digoxigenin-labelled riboprobes were then synthesized using this PCR product as a template. The digoxigenin-labelled riboprobes were then used in non-radioactive in situ hybridization to detect messenger RNA for interleukin-2 in mitogen stimulated peripheral blood mononuclear cells. This methodology has the potential for widespread application in immunology and cytokine research.
Birk
P E
PE
Department of Pediatrics, Children's Hospital of Winnipeg, Manitoba, Canada.
Grimm
P C
PC
D001483:Base Sequence; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D007376:Interleukin-2 / Q000235:genetics*; D008214:Lymphocytes / Q000187:drug effects; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015347:RNA Probes / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000302:isolation & purification*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007376:Interleukin-2; D015347:RNA Probes; D012333:RNA, Messenger; D013755:Tetradecanoylphorbol Acetate