normalization data transformation protocol
Results from microarray hybridization were analyzed using the Bioconductor package in R. Data were normalized with the “rma” procedure using a custom HGU133Plus2 annotation, to avoid known problems associated with the affymetrix annotation -. The normalized data was then analyzed using the “affy” -and “limma” -packages in Bioconductor. ID_REF = VALUE = RMA
array scanning protocol
All arrays were scanned in the Affymetrix GeneChip Scanner 3000 and the raw analysis was performed with Affymetrix GeneChip Operating System (GCOS) 1.4.
The samples were ice quenched and combined with the hybridization cocktail (5 ul oligonucleotide B2 control, 15 ul 20X eukaryotic hybridization controls, 150 ul 2X hybridization buffer, 30 ul 100% DMSO and 70 ul water). After 10 minutes of pre-hybridizing Human U133 Plus 2.0 Genome array at 45ºC, 60 rpm, 200 ul of cocktail was loaded onto each array and the arrays were hybridized for 16 hours at 45ºC, 60 rpm. The cocktail was removed and the arrays were stained and washed using the Affymetrix GeneChip Fluidics Station 450 and FS450_001 fluidics script.
One ug total RNA was combined with 2 ul T7 oligo(dT) primer and 2 ul Poly-A controls and brought to a volume of 12 ul. The samples were incubated at 70ºC for 10 minutes. A master mix of 4 ul first strand buffer, 2 ul DTT and 1 ul 10 mM dNTPs was added to the samples, followed by a two minute incubation at 42ºC. One ul Superscript II (Invitrogen, Carlsbad, CA) was added to each sample and the samples were incubated at 42ºC for one hour for first strand cDNA synthesis. A master mix of 91 ul water, 30 ul 5X second strand buffer, 3 ul 10 mM dNTPs, 4 ul DNA polymerase I, 1 ul E.coli DNA ligase, and 1 ul RNase H was added to each sample and the samples were incubated for two hours at 16ºC for second strand cDNA synthesis. Two ul of T4 DNA polymerase was added followed by a five minute incubation at 16ºC. Ten ul, 0.5 M EDTA was added to stop the reaction. cDNA clean-up was performed with the Affymetrix GeneChip Sample Clean-up Module, as per manufacturer’s instructions. The cDNA was eluted in 14 ul elution buffer. The final elution volume (~12ul) was combined with 28 ul of IVT mix master mix (4 ul 10X IVT labeling buffer, 12 ul Labeling NTP mix, 4 ul labeling enzyme mix, and 8 ul RNAse-free water) for the in vitro transcription cRNA synthesis reaction. The samples were incubated overnight for 16 hours at 37ºC followed by a hold at 4ºC. Clean-up was performed as per the manufacturer’s protocol using the Affymetrix GeneChip Sample Clean-up Module. The final elution volume was ~19ul. Concentration was checked via a nanodrop spectrophotometer. Six ul 5X fragmentation buffer was then combined with 15 ug cRNA and incubated at 95ºC for 35 minutes to fragment the cRN
nucleic acid extraction protocol
RNA was extracted and isolated from each sample separately using the Agencourt® RNAdvance Cell v2 system (Beckman Coulter Genomics, Danvers, MA); this extraction platform relies on paramagnetic bead purification of nucleic acids. It was linearly amplified using the NuGEN Ovation Pico WTA (linear amplification) System® (NuGEN Technologies, Inc., San Carlos, CA)
sample treatment protocol
H&E and trichrome stained sections were then graded and staged (Ishak fibrosis and Metavir) by a hepatopathologist who was blinded to all clinical information.
Tissues were divided into two at the time of biopsy. One piece was immediately placed in OCT and snap-frozen in liquid nitrogen.