strain: Cx3cr1GFP/+ tissue: Heart age: 2-3 month condition: 30 days post cryoinjury
Treatment protocol
Cryoinjury was used as myocardial infarction model. Animals were randomly sacrificed by CO2 inhalation 3, 7 and 30 days post-MI. Samples from healthy animals were not subjected to any surgery and were included as control.
Extracted molecule
total RNA
Extraction protocol
Total RNA,including small RNAs, was isolated from two-three independent biological replicates using miRNeasy Micro Kit with RNeasy MinElute Spin Columns (Qiagen) with DNaseI treatment.
Label
Cy3
Label protocol
miRNA from 100ng total RNA was Cyanine-3 (Cy3) labeled by miRNA Complete Labeling and Hyb Kit (Cat#5190-0456, Agilent technologies, Santa Clara, CA, US) following 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 55ºC, 20rpm for 20 hours according to the manufacturer’s instructions, hybridization section. After hybridization, slides were washed 5 minutes at room temperature with GE Wash Buffer 1 (Cat#5188-5327, Agilent technologies, Santa Clara, CA, U) and 5 minutes at 37ºC with GE Wash Buffer 2 (Cat#5188-5327, Agilent technologies, Santa Clara, CA, U).
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
Data processing
The scanned images were analysed with Feature Extraction Software (Agilent technologies, Santa Clara, CA, US) to obtain background substracted and spatially detrended Processed Signal intensities.
