strain: Sprague-Dawley gender: male tissue: renal cortex agent: L-mimosine
Treatment protocol
kidney tissue was dissected in ice-cold PBS to, remove medulla, and then snap-frozen in liquid nitrogen before transferring to storage at -80°C until further analysis
Growth protocol
All experiments were performed in male Sprague-Dawley rats weighing 180 to 200 g obtained from the Animal Centre, Shanghai Medical College, Fudan University (Shanghai, China). Rats were allowed free access to water and rodent food. All protocols were approved by the Institutional Animal Care Use Committee of Fudan University
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted and purified using RNAqueous® Kit(Cat#AM1912, Ambion, Austin, TX, US) | mirVana™ miRNA Isolation Kit(Cat#AM1560, Ambion, Austin, TX, US)| mirVana™ PARIS™ Kit(Cat#AM1556, Ambion, Austin, TX, US)following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100(Agilent technologies, Santa Clara, CA, US)
Label
Cy3
Label protocol
miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb Kit(Cat#5190-0456, Agilent technologies, Santa Clara, CA, US)followed the manufacturer’s instructions, labeling section.
Hybridization protocol
Each slide was hybridized with 100ng Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit(Cat#5190-0456, Agilent technologies, Santa Clara, CA, US)in hybridization Oven(Cat#G2545A, Agilent technologies, Santa Clara, CA, US)at 55℃，20rpm for 20 hours according to the manufacturer’s instructions, hybridization section. After hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US).
Scan protocol
Slides were scanned by Agilent Microarray Scanner(Cat#G2565BA，Agilent technologies, Santa Clara, CA, US)and Feature Extraction software (Agilent technologies, Santa Clara, CA, US)with default settings.
Description
microRNA expression after L-mim treatment in remnant kidney cortex
Data processing
Raw data were normalized by Quantile algorithm, Gene Spring Software (Agilent technologies, Santa Clara, CA, US).
