The RPE cells with 10 µmol/l 7-MX for 0, 24, 48, and 72 h were harvested and washed with PBS. Total RNA was isolated with TRIzol (Invitrogen Inc.), according to the manufacturer’s instructions. The quality and quantity of total RNA were estimated spectrophotometrically. Subsequently, RNA was reverse-transcribed into cDNA using a RevertAid First Strand cDNA synthesis kit (Fermentas, Burlington, Canada). Quantitative PCR (qPCR) was performed as described in a previous study [21 (link)]. Reactions were incubated at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The primers and probes for A1R (Rn00567668_m1), A2aR (Rn00583935_m1), A2bR (Rn00567697_m1), and GAPDH (Rn01775763_g1) were obtained from Applied Biosystems (Grand Island, NY). The amplification was performed in an ABI 7500 Fast Real-Time PCR system (Applied Biosystems). The 2-ΔΔ Ct method was used for relative quantification of the gene expression.
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Quantitative Analysis of Adenosine Receptor Expression
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Other organizations : First Affiliated Hospital of Chongqing Medical University, Chongqing Medical University, Sun Yat-sen University
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Variable analysis
independent variables
- Incubation time with 7-MX (0, 24, 48, and 72 hours)
- Gene expression levels of A1R, A2aR, and A2bR
- RPE cells
- Concentration of 7-MX (10 µmol/l)
- Wash with PBS
- Total RNA isolation using TRIzol
- RNA quality and quantity estimation
- Reverse transcription to cDNA using RevertAid First Strand cDNA synthesis kit
- Quantitative PCR (qPCR) with specific primers and probes for target genes and GAPDH
- Reaction conditions: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 15 s at 95 °C and 1 min at 60 °C
- ABI 7500 Fast Real-Time PCR system for amplification
- 2^(-ΔΔCt) method for relative quantification of gene expression
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