After approval of the local ethics committee and informed consent forms form the patients were obtained, patient fibroblasts with Tay-Sachs and Sandhoff were isolate from skin patient’s biopsies. They were obtained according to the Helsinki Declarations of 1964, as revised in 2001. Skin biopsies were placed in 10ml of transport media (PBS1X and antibiotics) and transported at RT. Individual samples were transferred to a sterile 35mm Petri dish and cut in a 5-10mm2 piece of tissue. Then, it was dissociated with forceps and scalpel transferring to a new 35mm Petri dish with the dermis upside down. The dish was placed inside the culture hood during 5-10 minutes to let the pieces of tissue dry and attach to the dish surface. After that, the samples were covered with DMEM culture 20% FBS and placed in the incubator 37ºC and 5% of CO2 until the fibroblast were propagated. Fibroblasts were cultured in high glucose media DMEM (Dulbeccoo`s modified media) (Gibco, Invitrogen, Eugene, OR, USA) supplemented with 20% fetal bovine serum (FBS) (Gibco, Invitrogen, Eugene, OR, USA) and 1% antibiotics (Sigma ChemicalCo., St Louis, MO, USA). Cells were incubated at 37ºC in a 5% CO2 atmosphere.
Extracted molecule
total RNA
Extraction protocol
Total RNA from fibroblasts were extracted using the RNeasy kit (RNeasy, QIAGEN, Valencia, CA) and then the quality of total RNA for array analysis was determined using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol
Hybridization protocol
100 ng of total RNA was hybridized to 5.2 μg of labelled cDNA
Scan protocol
GeneChips were scanned using the Affymetrix Scanner 3000 7G
Description
Gene expression data from healthy patient
Data processing
Array data were processed using Affymetrix® Genechip® Command Console® 2.0 with respect to background subtraction and normalization. We used the Affymetrix Microarrays ClariomTM D Arrays Human and Mouse, which includes 65956 genes. The raw data were analysed by the Affymetrix software Trancriptome Analysis Console (TAC) which analyse and visualize global expression patterns of genes, exons, pathways, and alternative splicing events.
