strain: B10.BR (H2-Kk) mouse as donor and B10.D2 (H2-Kd) mouse for recipient tissue: liver gender: male
Treatment protocol
B10.BR mice were used as donors and B10.D2 mice were used as recipients. Liver allo-transplantation surgery on the mice was performed in this combination. Three mice from each group were sacrificed, and the liver grafts were removed on days 5, 8, 14 and 100 after transplantation.
Growth protocol
All mice were maintained under standard conditions and fed rodent food and water, in accordance with the guidelines of the Animal Use and Care Committee of the National Research Institute for Child Health and Development.
Extracted molecule
total RNA
Extraction protocol
Total RNA, including miRNA from RNAlater (Ambion, Austin, TX) immersed liver grafts, was isolated using the RNeasy Mini Kit and miRNeasy kit (QIAGEN Inc., Valencia, CA.), according to the manufacturer’s protocol. The quality and quantity of the RNA were evaluated using the NanoDrop 1000 (Thermo Scientific Inc., Waltham, MA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
One hundred ng of total RNA was dephosphorylated with calf intestinal alkaline phosphatase (Takara Bio Inc., Shiga, Japan), followed by denaturing with heat in the presence of dimethyl sulfoxide (SIGMA-Aldrich Co., St. Louis, MO). A cyanine dye, cyanine3-cytidine bisphosphate (Agilent), was then joined to the dephosphorylated single-stranded RNA using T4RNA ligase (Ambion). MicroBioSpin 6 columns (Bio-Rad Laboratories Inc., Hercules, CA) were used to remove any unincorporated cyanine dye from the samples.
Hybridization protocol
The purified labeled miRNA probes were hybridized to 8x15 K mouse miRNA microarrays (Agilent Technologies) in a rotating hybridization oven at 20 rpm for 20 hours at 55 °C. After hybridization, the arrays were washed in Gene Expression Wash Buffer 1 with Triton X-102 followed by Gene Expression Wash Buffer 2 with Triton X-102.
Scan protocol
All slides were immediately scanned at a resolution of 5 µm with Green-PMT XDR Hi 100%, Lo 5% using an Agilent DNA Microarray Scan (Agilent). The resulting images were quantified using Agilent’s Feature Extraction software program. The differentially expressed miRNAs were identified using a standard protocol developed for miRNA gene arrays. To increase the reliability of the data and ease of detection, miRNA species with a hybridization intensity <1.5 times the average hybridization intensity (mean) were excluded from the analysis.
Description
Gene expression after 5day in syngrafting liver
Data processing
A miRNA clustering analysis was performed with the Hierarchical clustering algorithm provided in the Avadis, ArrayAssist 4.3 software package.