Title: Affymetrix CEL analysis. Description:
Affymetrix Generic Hybridization
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
GeneSpringGX expression analysis software, Agilent, version GS10
Human trabecular meshwork cells cells were scraped from tissue culture dishes with guanidine thiocyanate buffer (RLT, Qiagen). Total RNA was extracted by loading the solution onto a QIA ShredderTM column (Qiagen) and continued by the use of the RNeasy Mini kit with on-column RNase-free DNAse digestion according to manufacturer’s recommendations (Qiagen). Purified RNA was eluted in 30 ul RNase-free water and the concentration measured with a NanoDrop ND-100 spectrophotometer (Thermo Fisher Scientific). Total RNA recoveries averaged 65.4 ± 3.6 ug and 11.9 ± 1.1 ug per 10 and 3 cm culture dishes respectively. RNA quality was assessed by measuring the size distribution on an Agilent Bioanalyzer (Agilent Technologies). (Parameters: Extracted product = total_RNA, Amplification = none)
Human trabecular meshwork primary cells at passage 4, seeded on either 3 cm or 10 cm dishes, were grown to between 65-90% confluency, washed twice with PBS and exposed to recombinant adenoviruses in 1 ml or 3 ml serum-free medium respectively at multiplicity of infections (moi) ranging from 1.6 X103-1.6 X104 vg/cell. After exposure to the virus for 90 min, complete media was added and incubation continued for 48 h or 72 h.
Primary human trabecular meshwork (HTM) HTM-72 and HTM-137 cell lines were generated respectively from the trabecular meshworks dissected from residual cornea rims of 29 and 39 years old donors (North Carolina Eye Bank) after surgical corneal transplants at the University of North Carolina Eye Clinic. The tissue was cut into small pieces, carefully attached to the bottom of the 2% gelatin-coated 35 mm dish, and covered slipped with a drop of MEM Richter’s Modification medium (IMEM, HyClone) supplemented with 20% fetal bovine serum (FBS), 50 ug/ml gentamicin (Invitrogen). Cells from these specimens were not treated with enzymes and were allowed to grow from the explant for a period of 4 weeks changing the media every other day; upon confluency, cells were harvested and stored in liquid nitrogen. When reconstituted, these primary nontransformed cells are grown in IMEM, 10% FBS, gentamicin and subsist for seven to eight passages. In this experiment study all cells were used at passage 4. These outflow pathway cultures comprise all cell types involved in maintaining resistance to flow. That includes cells from the three distinct regions of the trabecular meshwork plus cells lining the Schlemm’s canal. Because most of the cells in these cultures come from the trabecular meshwork, they are commonly referred to as “trabecular meshwork cells”. (Parameters: start time = 48, time unit = hours, min temperature = 37, temperature unit = C, media = MEM Richter’s Modification medium (IMEM))