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Relative quantitative analysis of glucagon receptor mRNA in rat tissues

par Svoboda, Michal ;Tastenoy, Michèle ;Vertongen, Pascale ;Robberecht, Patrick
Référence Molecular and cellular endocrinology, 105, 2, page (131-137)
Publication Publié, 1994

Article révisé par les pairs

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Résumé :

Total RNA prepared from nine rat tissues were analyzed for their content in glucagon receptor mRNA by two independent hybridization approaches: (1) simple dot blot analysis using labelled oligodeoxynucleotide; (2) highly specific RNase protection assay using labelled antisense RNA. Hybridization signal was quantified by laser densitometric scanning of autoradiographies. Results were expressed for each method relative to the liver content (100%) for either a constant amount of total RNA or for a constant amount of β-actin mRNA. We obtained similar relative values of glucagon receptor mRNA per constant amount of total RNA by the two hybridization methods: in liver (100 and 100), in kidney (38 and 34), and in heart (12 and 11). The glucagon receptor mRNA was overestimated by the less specific dot assays, in adrenal glands (21 versus 10) and in adipose tissues (24 versus 5). In the stomach, brain, duodenum and lung, the signal was equal to or below the reliable quantification limit. Reverse transcription and polymerase chain reaction (RT-PCR) of glucagon receptor mRNA with limited cycle number were performed using two sets of primers: the first set amplified a single band at the 3' coding end, and the second, 3-6 bands at the 5' coding end, revealing tissue-specific polymorphism. RT-PCR data confirmed the presence of glucagon receptor mRNA in liver, kidney, heart, adrenal glands and adipose tissue, and allowed the detection of a very low amount of glucagon receptor mRNA in the stomach, the duodenum and brain but not in the lung. Although a simple dot hybridization assay could be used for analysis of glucagon receptor mRNA level in liver, heart and kidney, the more sophisticated RNase protection assay is necessary to obtain reliable results in tissues with a lower expression of messenger. The glucagon receptor presented high, tissue-specific mRNA polymorphism. This may contribute to the difference of the expression of the glucagon receptor in different tissues.