RNA was prepared using the RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) following the manufacturer's recommendations 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
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the Low Input Quick Amp Labeling Kit, One-Color (Cat.# 5190-2305, Agilent technologies, Santa Clara, CA, US) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA).
Hybridization protocol
Each slide was hybridized with 1.65ug Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat.# 5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat.# G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours 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), followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=5μm, PMT 100%, 10%, 16bit.
Description
Prss54ko1L testis
Data processing
Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, GeneSpring Software 12.6.1 (Agilent technologies, Santa Clara, CA, US).
