Sureclean plus purification kit

The products of our α-SMA and ß-actin specific real-time PCRs amplifying cDNA samples derived from the kidneys of mice underwent UUO were purified by SureClean Plus purification kit (Bioline, Taunton, MA, USA) and sequenced using BrightDye Terminator Cycle Sequencing Kit (Nimagen, Nijmegen, The Netherlands) according to the instructions of the manufacturer. Sanger sequencing was performed on ABI 3500 sequencer (Thermo Fischer Scientific, Waltham, MA, USA) and chromatograms were analyzed by Unipro UGENE software version 1.16.1. (UniPro, Novosibrisk, Russia).

The protocols for DNA extraction, amplification and sequencing followed Mantelatto et al. (2009) and Pileggi and Mantelatto (2010) .
An ~700-bp region of a partial sequence of the mitochondrial COI gene was amplified by the polymerase chain reaction (PCR) using the pair of primers: HCO1 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) and LCO1 (5’-GGTCAACAAATCATAAAGATATTGG-3’) (Folmer et al. 1994 ). The PCR reaction was performed in an Applied Biosystems Veriti® 96-well thermocycler, using the following thermal cycle: initial denaturing for 2 min at 94 °C followed by 35 denaturing cycles at 94 °C for 30 s, primer annealing at 50–58 °C for 30 s and extension at 72 °C for 1 min, and a final extension for 5 min at 72 °C. The PCR products were purified using the SureClean Plus® purification kit (Bioline) and were sequenced with the Big Dye® Terminator Cycle Sequencing kit in an ABI 3100 Genetic Analyzer® (Applied Biosystems Life Technologies). All sequences were confirmed by sequencing both strands.

Artificial templates of mouse α-SMA (mα-SMA_T), ß-actin (mß-actin_T), γ-cyto-actin (mγ-cyto-actin_T) and γ-smooth-actin (mγ-smooth-actin_T) covering all of the annealing sections of the examined primers were synthetized as gBlocks Gene Fragments by Integrated DNA Technologies (Coralville, IA, USA). Human and rat α-SMA (hα-SMA_T and rα-SMA_T) and ß-actin (hß-actin_T and rß-actin_T) DNA templates were synthetized by PCR method using specific human or rat α-SMA and ß-actin primers. RT-PCR products were then separated by electrophoresis in 2% agarose gel. Thereafter, fractions with the required product length were extracted from the gel, purified by SureClean Plus purification kit (Bioline, Taunton, MA, USA) and resolved in RNase-free water.