normalization data transformation protocol
The raw data files extraced from Agilent Feature extraction software are processed and analyzed by GeneSpring GX11 software. ID_REF = VALUE = Normalized signal intensity
array scanning protocol
Microarrays were scanned with Agilent Technologies Scanner (G2505C US45102880) at 5um resolution at 100% PTM. The images were then imported into Agilent Feature Extraction Software.
1.65ug labeled with Cy3 for each sample was fragmented, denatured and then hybridized to Agilent 4x44 rat whole genome micrarray slides for 17 hours at 65C. Slides were then washed according to the protocol.
Following Agilent One-Color Microarray-Based Gene Expression protocol of the Low RNA Input Fluoresent Linear Amplification Kit Plus (Agilent, Santa Clara, CA), 200ng RNA with the addtion of One-Color Spike Mix was denatured and incubate with T7 Promoter perimer. Synthesis of cDNA followed with the addtion of First-Strand buffer, DTT, dNTP mix, MMLV-RT and RNAse Out and incubation at 40C for 2 hours. Transcription of the product incorporated the Cyanine 3-CTP in the master Mix with incubation at 40C for 2 hours.
nucleic acid extraction protocol
Fresh ECT samples were homogenized by an Omnitip Tissue homogenizer (Cat. No.: 6615-7273, USA Scientific, Ocala, FL, USA). Total RNAs were isolated using Invitrogen Trizol and purified by RNeasy® Mini Kit (Qiagen, Cat. No.: 74104). Then the RNA quality and quantity were measured using the NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the Bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA, USA).
sample treatment protocol
To determine the effect of cyclic mechanical stretch and/or p38MAPK inhibition on ECT gene changes, we exposed culture day-5 ECT to uniaxial cyclic mechanical stretch (0.5 Hz and 5% elongation); to p38MAPK inhibition by BIRB796 (Catalog No. S1574, Selleck Chemicals, 10µM), or both stretch and BIRB796.
Approximately 200 µL of cell/matrix mixture, containing approximately 1x106 cells, was poured into the trough and incubated for 150 min in a standard CO2 incubator (37°C, 5% CO2) to form a cylindrical construct. Both ends of the construct were held by anchors attached to the Tissue Train culture plate. When the tissue was formed, the culture plate was filled with a growth medium containing 10% fetal bovine serum (Invitrogen), 1% antibiotic–antimycotic solution (Atlanta Biologicals), and 1% chicken embryo extract. Constructed engineered tissues were cultured for 7 days and the culture medium was changed every other day