Adult male Sprague-Dawley rats were randomly assigned to two groups: injured and control. Five animals per time point per group were used for gene expression analysis and an additional animal from the sham and injury group at each time point was fixed in formalin and evaluated histologically following paraffin embedding and hematoxylin and eosin stain.. After receiving buprenorphine subcutaneously 15 minutes prior to surgery (0.1 mg/kg), the injured group underwent general anesthesia for the surgery, Anesthesia was induced by placing rats into a Plexiglas chamber flushed with 1-3% Isoflurane and oxygen and they were then maintained at 1-3% Isoflurane with a nose cone on a Bain circuit (with dead space-eliminating rodent anesthesia circuit) hooked to the rodent gas anesthesia machine (VetEquip, Inc., Pleasanton, CA). Respiration was monitored during this 10-20 minute procedure. After the surgery, analgesia (buprenorphine; 0.1 mg/kg SC every 12 hours) was administered for discomfort associated with the surgery for the first 72 hr and as needed. The sham control groups were exposed to the same analgesi/anesthesia regime and a bilateral sham surgery was performed but the ligament was not severed. Animals from each group were sacrificed sequentially on the following schedule: Day 1, 2, 4, 7, 10, and 14. Tissue from the MCLs were harvested under anesthesia and frozen in liquid nitrogen prior to RNA isolation or fixed in 10% formalin and then euthanasia was performed. A group of five rats that were not manipulated any way served as controls for both sham and injured rats. Eight ligaments were treated with 0.1% collagenase in Delbecco's Modified Eagle's Medium (DMEM: Invitrogen, Carlsbad, CA)) for 3 h, rinsed in complete medium (10% fetal bovine serum; FBS (Hyclone) in DMEM and cultured for two weeks until confluent. Immunostaining for rat proyl hydroxylase confirmed the fibroblast origin of these cells. Rat ligament fibroblasts (RLF) were subcultivated with 0.1% Trypsin/EDTA (Invitrogen) into 6-well multiplates (Corning, Ithaca, NY) until 90% confluent RNA was isolated with TRI™ Reagent as for ligaments.
Growth protocol
Five animals per time point per group were used for gene expression analysis and an additional animal from the sham and injury group at each time point was fixed in formalin and evaluated histologically following paraffin embedding and hematoxylin and eosin stain.. After receiving buprenorphine subcutaneously 15 minutes prior to surgery (0.1 mg/kg), the injured group underwent general anesthesia for the surgery, Anesthesia was induced by placing rats into a Plexiglas chamber flushed with 1-3% Isoflurane and oxygen and they were then maintained at 1-3% Isoflurane with a nose cone on a Bain circuit (with dead space-eliminating rodent anesthesia circuit) hooked to the rodent gas anesthesia machine (VetEquip, Inc., Pleasanton, CA). Respiration was monitored during this 10-20 minute procedure. After the surgery, analgesia (buprenorphine; 0.1 mg/kg SC every 12 hours) was administered for discomfort associated with the surgery for the first 72 hr and as needed. The sham control groups were exposed to the same analgesi/anesthesia regime and a bilateral sham surgery was performed but the ligament was not severed. Animals from each group were sacrificed sequentially on the following schedule: Day 1, 2, 4, 7, 10, and 14. Tissue from the MCLs were harvested under anesthesia and frozen in liquid nitrogen prior to RNA isolation or fixed in 10% formalin and then euthanasia was performed. A group of five rats that were not manipulated any way served as controls for both sham and injured rats.
Extracted molecule
total RNA
Extraction protocol
Prior to total RNA isolation, all ligaments were lyophilized. The dried ligaments were minced with a scapel blade and placed in 1 ml of TRI™ Reagent. The process yields 0.5-3 µg per specimen, the injured specimens yielding more RNA. The resulting RNA was quantified on a Nanodrop spectrophotometer and quality assessed by gel electrophoresis.
Label
Cy3
Label protocol
Microarray analysis was performed using the Agilent (Santa Clara, CA) low-input labeling system and the Cy-3 or Cy-5 labeled RNA applied to the rat whole genome chip (Agilent).
Hybridization protocol
750 ng of labeled, amplified cRNA was fragmented with according to Agilent's procotol at 60 degrees for 30 and 2X hybridization buffered added. Hybridization was accomplished with the Agilent hybridization chamber in a Robbin's Scientific oven for 17 hrs.
Scan protocol
Microarray image segmentation analysis was performed with an Axon 4000B and GenePix 6.0 software and the resulting Cy-3 and Cy-5 intensities recovered from each ligament for further analysis.
Description
Intensity data plus the flags are highlighted in yellow in each raw data (.gpr) file.
Data processing
The Cy 3 and Cy5 pixel intensities minus background were extracted and placed in a single flat file and each microarray sample treated as a single dye labeled specimen. Statistical analyses were performed using BRB ArrayTools developed by Dr. Richard Simon and Amy Peng Lam.
Expression Profiling of Severed Rat Medial Collateral Ligament Following Injury
Data table header descriptions
ID_REF
VALUE
Statistical analyses were performed using BRB ArrayTools developed by Dr. Richard Simon and Amy Peng Lam. Global normalization was used to median center the log-ratios on each array in order to adjust for differences in labeling intensitites of the Cy3 and Cy5 dyes {Simon, 2003 #1885}. No filtering was performed and the only requirement for inclusion was achieving a statistically significant difference between time points.
