Primary human hepatocyte cultures were transfected with genomic RNAs of HCV genotypes 1a, 1b, and 2a (1 μg/106 cells) using FuGENE6 (Roche). On day 6 postinfection, the small RNA (≤200-nucleotide) fraction was enriched from HCV-infected cell RNA using a mirVana isolation selleckchem kit (Ambion). Four micrograms of each sample together with positive control (synthetic Arabidopsis thaliana mir-157a, which is not present in the human genome) was spiked in and was hybridized to the microarray slide (BioMicro
System). After 16 hours, the hybridized microarray was washed with a standard sodium citrate solution to remove unhybridized probes. After 3 hours of Klenow exonuclease-1 incubation, exo(-) Klenow enzyme was added to extend the miRNAs hybridized to the chip-attached templates in a primer extension step. During this step, biotinylated dATP was
incorporated as a final portion of the extension through the designed polythymidine region. Detection of this template-hybridized miRNA was performed using streptovidin-conjugated Alexa-fluor-555, which binds to the biotinylated stretch of A’s at the 3′-end of the captured miRNA. Fluorescence data sets were collected using GenePix 4000 scanner (Axon). Details of the procedure are described in Yeung et al.14 Primary hepatocytes were transfected with HCV1a genomic RNA (1 μg/106 cells) in triplicate. Parallel cultures were transfected with DLC-1 complementary DNA (cDNA) expression vector (50 ng/106 cells for 6 hours) prior to transfection with HCV 1a genomic RNA. Six days posttransfection, the cells were released with 0.05%
trypsin treatment and were resuspended at 104/100 μL in (phosphate-buffered selleck chemicals llc saline containing 2% fetal bovine serum) processed for Ki67 immunostaining (BD Biosciences) according 上海皓元 to the manufacturer’s instructions. Primary human hepatocytes were transfected with HCV genotypes 1a, 1b, and 2a (1 μg/106 cells) as described.12 Virus released in the culture medium was filtered through 0.25-μm filters from infected cells.12 Viral RNA replication was evaluated at indicated times after infection as outlined above, and the efficiency of virus released in the culture media was validated using the World Health Organization’s HCV standards (Acrometrix, Benicia, CA). Primary human hepatocyte culture was cotransfected with luciferase reporter containing DLC-1 3′ untranslated region (UTR) (50 ng/106 cells), miR-141 (50 nM/106 cells, antagomir) or miR-141 (50 nM/106 cells, Mimic) using Lipofectamine 2000 (Invitrogen). Luciferase assays (Promega) were performed on the third day after transfection according to the manufacturer’s instructions. The results are given as the mean ± SE. Statistical analysis of the data was performed using the Student t test, Fisher’s exact test, or otherwise as described. To assess virus infection-associated changes in host gene expression, we analyzed alterations in miRNAs in primary human hepatocytes infected with HCV genotypes 1a, 1b, and 2a (Supporting Information Fig. 1).