6 protocols
array scanning protocol
The data from scanned microarray images were extracted using Agilent Feature Extraction software. Resulting ratio for each locus is averaged of replicated spots and used for further analysis.
Hybridization was performed according to Agilent aCGH array hybridization protocol.
MCA experimet sample after MCA were labeled with Cy3 and Cy5 using a random prime labelling kit (Invitrogen, Carlsbad, California)
Genomic DNA was extracted using a standard phenol chloro for MP Method. 2ug of genomic DNA was digested with 100 U of methylation-sensitive restriction endonuclease SmaI (New England Biolabs), which cuts unmethylated DNA and leaves blunt ends (CCC/GGG). Subsequently, the DNA was digested with 50 U of methylation-insensitive restriction endonuclease XmaI, which leaves sticky ends (C/CCGGG). Adaptors were ligated to the sticky ends. The adaptors were prepared by incubation of the oligonucleotide RMCA12 (5�Œ-CCGGGCAGAAAG-3�Œ) and RMCA24 (5�Œ-CCACCGCCATCCGAGCCTTTCTGC-3�Œ) at 65 �C for 2 min, followed by cooling to room temperature for 60 min. Digested DNA was ligated to the adaptor using T4 DNA ligase (Invitrogen, Carlsbad, CA) and amplified by PCR using RMCA24 primer. The PCR condition was 5 min at 72 �C to fill in the overhanging ends of the ligated DNA fragments, and at 95 �C for 3 min; this was then followed by 25 cycles of 1 min at 95 C and 3 min at 77 �C, with a final extension of 10 min at 72 �C.
Huh7.5.1, were grown in DMEM(Invitrogen, Carlsbad, CA), plus 10% FBS in plastic tissue culture plates in a humidified atmosphere containing 5% CO 2 at 37�C.
Tissue culture-based HCV infection system in Huh7.5.1 was conducted using an HCV-JFH1 strain. (Wakita T et al, Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005;11:791-6.)