Quantitative analysis of rat thyroglobulin messenger RNA in FRTL-5 cells by competitive polymerase chain reaction with human thyroglobulin messenger RNA.
To measure relative expression level of mRNA in a small number of cultured rat thyroid cells (FRTL-5), we developed a system of a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Human thyroglobulin mRNA in total RNA extracted from a human thyroid tissue was used as an internal control. FRTL-5 cells in a 24 well dish were lysed with denaturing solution containing human RNA. Total RNA was extracted followed by reverse transcription and polymerase chain reaction. After digestion with a restriction enzyme, PCR products were separated by electrophoresis and stained with Sybr Green I, then their fluorescence was measured with fluorescent image analyser. Increase of thyroglobulin mRNA in FRTL-5 cells stimulated by thyroid stimulating hormone (TSH) was observed by this technique. Because this method does not require a large number of cells or radioactive isotopes, it is as useful for the analysis of the relative expression level of mRNAs in the cells as the conservative methods such as Northern Blot.
Takano
T
T
Department of Laboratory Medicine, Osaka University Medical School, Japan.
Amino
N
N
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D006801:Humans; D015151:Immunoblotting; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D013268:Stimulation, Chemical; D013954:Thyroglobulin / Q000235:genetics*; D013972:Thyrotropin / Q000494:pharmacology; D014158:Transcription, Genetic
D012333:RNA, Messenger; D013972:Thyrotropin; D013954:Thyroglobulin