l samples. Furthermore, a “needle to freezer time” of less than 30 min was defined for all samples. All samples were handled at 4C until they were stored for final analysis at -80C. RNA extraction and cDNA transcription were performed simultaneously for all samples. The study was approved by the Ethics Committee of Faculty of Pharmacy, Cairo University. Sixty-seven consecutive outpatients with ages ranging from 20 55 years with chronic HCV-4 and twenty-seven healthy volunteers were included in the prospective study. Written informed consent, approved by the Ethics Committee of Faculty of Pharmacy, Cairo University, was obtained from all subjects in this study. Chronic HCV was diagnosed at the SAR 405 manufacturer Tropical Medicine and Hepatology Department of El-Kasr El Aini Hospital, and patients were referred for antiviral therapy with PEG-IFN/RBV. Blood samples were collected from all patients prior to the start of treatment for analysis of all biochemical markers, HCV viral loads, genotyping, and miRNA estimation. Subsequently, the HCV genotype-4 patients were divided depending on their responses to antiviral therapy into 45 sustained virological responders, for whom viral RNA remained undetectable 6 months after the cessation of the 6 month treatment period, and 22 non-responders, for whom viral RNA remained detectable after 6 months of treatment. HCV Genotyping using the Nested-PCR/Ohno Method RNA was isolated from sera for HCV genotyping using a Qiagen Viral RNA kit. HCV-RNA genotyping was performed using the Ohno method, which relies on nested PCR amplification of the virus core 
gene using genotype-specific primers as previously described. Assessment of HCV-4 RNA Loads by Real-Time PCR Viral loads were measured by real-time reverse transcription polymerase chain reaction using a Light Cycler system after RNA extraction from sera using a Qiagen Viral RNA kit. Amplification primers for HCV were: 5′ primer K78F and 3′ primer 3 / 12 MicroRNAs as Predictor Markers for Response to Treatment in HCV K80R. The hybridisation probes FL 5’3′ and LC 5’3′ were used to detect the product. HCV-RNA in serum was measured before treatment and routinely at weeks 24 and 48 after treatment and graded into low, moderate and high levels. Biochemical Investigations Blood samples were collected from all volunteers and immediately centrifuged at 4C; the serum supernatants were frozen at -20C. All analyses were performed in duplicate. Laboratory tests including ALT, AST, alkaline phosphatase, total bilirubin, direct bilirubin, albumin, creatinine and -fetoprotein as well as complete blood counts including haemoglobin and total leukocyte counts were performed in the HCV genotype-4 infected patients treated with PEG-IFN/RBV antiviral therapy and healthy controls. Serum miRNA Assay. Total RNA with preserved miRNAs was extracted from 200 l serum with the miRNeasy extraction kit using 1 ml QIAzol lysis reagent and incubated for 5 min at RT. Then, 200 L of chloroform was added, and the samples were vortexed for 15 sec, and incubated for 23 min at room temperature. This was followed by centrifugation at 14,000 xg at 4C for 15 min. The upper watery phase was removed, and an equal volume of 100% ethanol was added. Each 700 l of this mixture were placed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19762901 in miRNeasy Mini spin column in a 2 ml collection tube and centrifuged at PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763758 8000 xg at room temperature for 15 sec. After the mixture completely passed through the column, 700 l of buffer RWT was added to each column prior to centrifugatio