gender: male age: 48 ishak fiborosis score: not applicable
Extracted molecule
total RNA
Extraction protocol
We extracted RNAs from plasma samples in 16 healthy controls, 16 CHC patients with mild fibrosis and 16 with significant fibrosis. Total RNAs including miRNAs in plasma were purified with miRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany) respectively, following the manufacturer’s instructions.
Label
biotin
Label protocol
Total RNA derived from 400 μL of plasma in each subject was poly(A) tailed and then directly ligated to a fluorescent dendrimer (a branched single and double-stranded DNA molecule conjugated to biotin) using the FlashTag™ Biotin HSR RNA Labeling Kit (Affymetrix), following the manufacturer’s instructions (User manual of the FlashTag™ Biotin HSR RNA Labeling Kit, Affymetrix)
Hybridization protocol
The biotin-labeled samples with hybridization master mix were hybridized at 48 °C and 60 rpm for 16 hours on Affymetrix GeneChip miRNA 4.0 Array, following the manufacturer’s instructions (User manual of the FlashTag™ Biotin HSR RNA Labeling Kit, Affymetrix) . Thereafter, the GeneChips were washed and stained in the GeneChip® Fluidics Station 450 (Affymetrix), following the manufacturer’s instructions.
Scan protocol
The GeneChips were scanned using GeneChip® Scanner 3000 7G (Affymetrix), following the manufacturer’s instructions.
Data processing
Raw microarray data (CEL files) were imported into Partek Genomics Suite (Partek Inc., St. Louis, MO), and probe set summaries were computed using Robust Multi-Array algorithm. To adjust for differences in labeling intensities and hybridization, a global normalization was made by aligning signal intensities of data arrays across the medians.
