BACKGROUND: Peripheral blood mononuclear cells and isolated polymorphonuclear neutrophilis were used to evaluate gene expression studies. Unfortunately, there are many methodological problems related to these cellular models, limiting their use. The aim was to evaluate a fast and easy procedure for the extraction of total RNA from leukocytes obtained from human whole blood (WB) < 10 mL; to determine adenosine receptor (AR) mRNA expression in WB samples of normal subjects and to establish the most stable reference genes for data normalization. METHODS:mRNA expression was performed by Real-Time PCR. RESULTS:The most stably expressed genes were TPT1, EEF1A, and RPL13A. Similar levels of mRNA expression were observed for A2aR, A2bR, and A3R while lower levels were measured for A1R (p = 0.02 A1R vs. A2aR; p = 0.04 A1R vs. A3R). CONCLUSIONS:Our study represents an important and useful starting point for future investigations devoted to evaluate the expression of ARs in human diseases.
Adenosine Receptor Expression and Gene Reference Evaluation in Human Leukocytes
Sabatino Laura;Cabiati Manuela;Caselli Chiara;Del Ry Silvia
2013
Abstract
BACKGROUND: Peripheral blood mononuclear cells and isolated polymorphonuclear neutrophilis were used to evaluate gene expression studies. Unfortunately, there are many methodological problems related to these cellular models, limiting their use. The aim was to evaluate a fast and easy procedure for the extraction of total RNA from leukocytes obtained from human whole blood (WB) < 10 mL; to determine adenosine receptor (AR) mRNA expression in WB samples of normal subjects and to establish the most stable reference genes for data normalization. METHODS:mRNA expression was performed by Real-Time PCR. RESULTS:The most stably expressed genes were TPT1, EEF1A, and RPL13A. Similar levels of mRNA expression were observed for A2aR, A2bR, and A3R while lower levels were measured for A1R (p = 0.02 A1R vs. A2aR; p = 0.04 A1R vs. A3R). CONCLUSIONS:Our study represents an important and useful starting point for future investigations devoted to evaluate the expression of ARs in human diseases.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
