Quantification of Endothelin-1 mRNA and Endothelin Receptor B mRNA from Cortex and Inner Medulla of Mouse Kidney Using Real-time RT-PCR

Ahn, Do Whan

Abstract

BACKGROUND
Endothelin-1 (ET-1) production and ET receptor type B (ETRB) density in the kidney increase along the corticomedullary axis. Northern blot showed that preproET-1 mRNA message was higher in medulla than cortex. ETRB mRNA expression by nested RT-PCR was similar to that of ET-1 mRNA. This study was undertaken to evaluate the renal expression pattern of ET-1 and ETRB mRNAs by Northern blot and nested RT-PCR using a real-time RT-PCR technique. METHODS: The cortex and inner medulla of mouse kidney were dissected out and real-time RT- PCR was performed. Fluorescent ntensity of PCR was recorded using the DNA-intercalating dye SYBR green. Melting curve analysis was utilized for selection of sequence-specific PCR products. For the comparision of relative expression of mRNA in the cortex and inner medulla, Pfaffl's formula (Pfaffl MW: Nucleic Acids Res 29: 2003-2007, 2001) was used. RESULTS: Peak Tms of amplified beta-actin, ET-1 and ETRB genes from inner medulla were 88.2degrees C, 90.5degrees C and 85.5degrees C, respectively. In the cortex, expression of ETRB mRNA was greater on an average by 4.59 cycles (24.59=24 times more abundant) compared to that of ET-1 mRNA. In the inner medulla, ETRB message was 25 times greater than ET-1 message. When ET-1 mRNA expression was compared between cortex and inner medulla the ratio was 3.2, indicating that inner medulla contained three times more ET-1 mRNA than cortex. The expression ratio of cortex to inner medulla of ETRB mRNA was 3.3. CONCLUSION: These results suggest that ET-1 mRNA and ETRB mRNA are more abundantly expressed in medulla than cortex and that the distribution of these mRNAs may be intimately related to that of their respective gene products.