2.4. Preparation of Total RNA and miRNA Microarray Analysis

The rat kidney tissues were stored in RNAlater Stabilization Solution (Thermo Fisher Scientific, PA, USA) at −80°C. Total RNA of the rat kidney tissues were extracted using the Trizol reagent and reverse transcribed to cDNA using the PrimeScript RT reagent kit according to the manufacturer's instructions. The quality of the extracted RNA was verified by measuring the A260/A280 ratio on a spectrophotometer and by agarose-formaldehyde gel electrophoresis. Approximately 5 μg of the RNA was size-fractionated using a YM-100 Microcon centrifugal filter (Millipore, MA, USA) to isolate fragments shorter than 300 nucleotides, which were then 3′-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail to allow subsequent fluorescent dye staining. Overnight hybridization was conducted using a micro circulation pump (Atactic Technologies, TX, USA) and a μParaflo microfluidic chip (LC Sciences, TX, USA) [22]. Following RNA hybridization, detection was performed by circulating tag-specific Cy3 dye through the microfluidic chip. Images were collected on a GenePix 4000B microarray scanner (LC Sciences) and digitized by the Array-Pro Analyzer software (Media Cybernetics, MD, USA). The results were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (LC Sciences) [23]. p < 0.05 was considered statistically significant.