gender: Male strain: SD surgery: TAC sample time after surgery: 5 days
Treatment protocol
The SD rats were subjected to transverse aortic constriction surgery. Rats were anesthetized with barbital sodium administrated intraperitoneally at 200 mg/kg body weight. An approximately two-centimeter incision was made at the level of the cricoid process and dissected down to the clavicle. After cutting the right clavicle, the thymus was retracted gently, and the aortic arch was exposed. A constriction of the aortic arch was conducted between both carotid arteries using an 18 gauge angiocatheter. Sham-operated rats were underwent a similar surgical procedure without constriction of the aorta. After TAC surgery, the chest was closed with a 5-0 nylon suture, and mice were placed on a heating pad at 37°C until they had completely recovered from anesthesia.
Growth protocol
SD male rats (6-8 weeks old, 200-300 g) were housed at 20±2°C and 55±20% humidity with 12-hr light/dark cycles and free access to food and water in the Animal Care Facility at the Sun Yat-sen University (SYSU) School of Medicine.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from hearts at 5, 10, 15 and 20 days after TAC or sham surgery using TRIzol (Invitrogen，Life Technologies，New York， USA) and an miRNeasy Mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. RNA quality and quantity were measured using a Nanodrop spectrophotometer (ND-1000, Thermo Fisher Scientific, Wilmington, DE, USA), and RNA integrity was determined by denaturing agarose gel electrophoresis.
Label
Hy3
Label protocol
After RNA isolation from the hearts, one microgram of each sample was 3'-end-labeled with a Hy3TM fluorescent label (Exiqon, Denmark) using T4 RNA ligase.
Hybridization protocol
After the labeling procedure, the Hy3TM-labeled samples were hybridized to a miRCURYTM LNA Array (v.16.0) (Exiqon),which contains more than 1,891 capture probes covering all of the human, mouse and rat miRs annotated in miRBase 16.0, as well as all viral miRs related to these species.
Scan protocol
The images on the chip were scanned and imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction.
Description
The miRNAs in Sham-operated rats were set up as the blank control.
Data processing
The 5 replicates were averaged, and the miRs that underwent a >= 2-fold change in expression between the TAC and sham groups were selected for analysisand.Meanwhile,the miRs with intensities > 50 in all samples were used to calculate the normalization factor. Expression data were normalized using the median normalization. After normalization, differentially expressed miRs were identified by fold change filtering (fold change >= 2). Hierarchical clustering was performed using MEV software (v4.6, TIGR).
