Cells were grown on DMEM medium (InVitrogen, Carlsbad, CA) supplemented with 20% heat-inactivated fetal bovine serum (Hyclone, Logan, UT) and 2 mM L-glutamine, and were used before passage 10. All cultures were initiated at 7.5 x 105 cells per 75 cm2 flask. The culture medium was changed every 3-4 days.
Extracted molecule
total RNA
Extraction protocol
On day 16, approximately 10 days post-confluence, cells were harvested following trypsinization. Cells from triplicate flasks were pooled, washed twice with phosphate buffered saline and re-suspended in PBS. Cell numbers were determined using a Coulter Counter Model Z. The total cell suspension was divided into aliquots for RNA extraction, lipid analysis, and determination of residual glucocerebrosidase activity. The cell suspensions were centrifuged at 1000 x g for 5 min and dry cell pellets were stored at -80 C until used.
Label
biotin
Label protocol
Labeling was performed on a sample-by-sample basis according to manufacturer’s guidelines for use with the Affymetrix Human Genome U133A_2 GeneChip (Affymetrix, Inc). DNase treatment was included as part of isolation to remove possible contaminating DNA. Bioanalyzer nanochip (Agilent, Inc) and NanoDrop (ThermoFisher, Inc) were used to validate and quantitate the RNA prior to cRNA synthesis and labeling. For cRNA synthesis and labeling, 5ug of total RNA was used per sample in conjunction with the Affymetrix 3’ one-cycle Target labeling Kit (Affymetrix, Inc).
Hybridization protocol
Labeled cRNA were hybridized to Affymetrix Human U133A_2 GeneChips (Affymetrix, Inc) in blinded interleaved fashion.
Scan protocol
Affymetrix scanner 3000 was used in conjunction with Affymetrix GeneChip Operation Software to generate ~580,000 probe measurements/hybridized cRNA.
The “rma” function in bioconductor (http://www.bioconductor.org/) was used to summarize probe measurements and generate normalized gene fragment expression values for each hybridized cRNA. Quality of data was assured via sample-level inspection by Tukey box plot, covariance-based PCA scatter plot, and correlation-based Heat Map. Gene fragments not having at least one expression value greater then system noise were deemed non-informative and discarded. System noise was defined as the lowest expression value at which the LOWESS fit of observed CV for each gene fragment by mean expression value for each gene fragment changes from non-linear to linear. For gene fragments not discarded, expression values were floored to equal system noise if less than system noise then subject to significance testing. The one-factor ANOVA under BH FDR MCC condition (alpha criteria<0.05) was used to identify gene fragments having expression values significantly associated with differences in gender. Spearman correlation under BH FDR MCC condition (alpha criteria<0.05) was used to identify gene fragments having expression values significantly associated with differences in age. Gene fragments found to have expression values significantly associated with differences in age and/or gender were discarded before testing gene fragment expression for significant association with Type I and/or Type III GD. Testing gene fragment expression for significant association with Type I and/or Type III GD was done using the one-factor ANOVA under BH FDR MCC condition (alpha criteria<0.05). For gene fragments found to have expression significantly associated with Type I and/or Type III GD, the Tukey HSD test was used to code gene fragments having a post-hoc p-value < 0.05 between Type I GD vs. control, Type III GD vs. control, and/or Type III GD vs. Type I GD. These codes were used classify gene fragments having expression significantly association with Type I and/or Type III GD as significantly dysregulated in Type I GD only, significantly dysregulated in Type III GD only, significantly and concordantly dysregulated in both Type I GD and Type III GD, or significantly and disconcordantly dysregulated in both Type I GD and Type III GD. Gene fragment annotations and associated gene functions were obtained using IPA (Ingenuity, Inc.).
