2.7 Quantification of Epo mRNA expression

Epo mRNA were quantified in kidney tissues using the qRT−PCR method (Chomczynski and Mackey, 1995). Firstly, the kidney (100 mg wet weight) was dissected, homogenized with TRIzol reagent (1 ml) and incubated at room temperature for 5 min. Secondly, chloroform (200 µl) was added to the reaction mixture, and it was then incubated at room temperature for 5 min and centrifuged at 12,000 g, 4°C for 15 min. Thirdly, the aqueous−phase layer was transferred to a new tube, mixed with isopropanol (500 µl), then incubated at room temperature for 5 min and centrifuged at 12,000 g, 4°C for 10 min. Fourthly, RNA (white pellets) was collected, centrifuged-washed with 75% ethanol at 7,500g, 4°C for 5 min and dried. It was then resuspended in DEPC water (20 µL). RNA concentration and purity values were determined by NanoDrop spectrophotometer using wavelengths of 260 and 280 nm.

In assay, total RNA (1 µg) was reversely transcribed to complementary DNA (cDNA) using a Thermo Scientific RevertAid First Strand cDNA Synthesis kit according to the manufacturer’s protocol and instructions. Then, cDNA concentration and purity values were measured with a microvolume spectrophotometer (NanoDrop™, Thermo Fisher Scientific, Waltham, MA, United States) using the wavelengths of 260 and 280 nm.

Accordingly, qRT−PCR analysis of cDNA was performed using a Maxima SYBR Green/ROX qPCR kit on the Applied Biosystems™ QuanStudio™ 6 Flex real−time PCR instrument according to the manufacturer’s instructions. The primer sequences used in this study are presented in Table 1. The instrument was set up with a default thermal cycler protocol provided by the producer as follows: 95°C for 10 min, 95°C for 15 s, 58°C for 30 s and 72°C for 30 s for 40 cycles. For each PCR reaction, 1,000 ng of cDNA was used as a template. All analyses were carried out in triplicate. Relative quantities were presented in each sample, which were then assessed with the 2^−(ΔΔCt) method.

List of primers used in qRT−PCR.

Abbreviations: Epo, erythropoietin gene; Erfe, erythroferrone gene; qRT−PCR, quantitative reverse-transcription polymerase chain reaction; Hprt, hypoxanthine-phosphoribosyltransferase gene.