��-actin AMN-107 was used for normalization. Evaluation of ��-actin expression was performed by the SYBER Green PCR. Briefly, one ��g of total RNA was subjected to the reverse transcription reaction using PrimeScript RT reagent Kit (Takara Bio Co., Shiga, Japan). Then, qRT-PCR was done using SYBR premix Ex Taq II (Takara Bio Co.). All values were corrected by each calibration curve and the relative expression level was measured using the ����Ct method. Primers for PCR are shown in Table S1. Luciferase Reporter Assay 0.5��g of each construct was transfected into 30~40% confluent DLD-1 cells that were cultured in 48-well plates w/wo 10 p mole of miRNA mimics. Seventy two hours after transfection, cells were subjected to Dual�CLuciferase Reporter Assay (Promega).

For normalization, a renilla luciferase vector, pRL-TK (Promega), was co-transfected. Statistics Significant difference from the control was analyzed by the Mann�CWhitney U test *: p<0.05, **: p<0.01. Supporting Information Figure S1 Genotyping of mice by PCR. A) PCR analysis of genomic DNA of the transgenic mice DNA extracted from mice tail was analyzed by PCR primers for the vector and human pri-miR-143(see Fig.1A). Ethidium bromide staining image of polyacrylamide gel is shown. The arrow indicates the transgenic allele. W: wild mouse, Tg: transgenic mouse B) PCR analysis of genomic DNA of APCMin/+ mice. DNA extracted from mice tail was analyzed by PCR primers for APCMin/+. The upper and lower arrows indicate the wild-type and the APCMin alleles, respectively. Ethidium bromide staining image of polyacrylamide gel is shown.

(TIF) Click here for additional data file.(710K, tif) Figure S2 Histological analysis of the small intestine tumors in transgenic mice. Tumors of the intestine in four month-old mice were stained with hematoxylin and eosin. A representative small intestine tumor of a non-transgenic W/APC mouse is shown at (A) 40 �� and (C) 200 �� magnification. A Anacetrapib representative small intestine tumor of its littermate Tg/APC mouse is shown at (B) 40 �� and (D) 200 �� magnification. Adenomatous polyps at similar differential stages developed in both mice. Black boxes indicated the areas shown in higher magnification. (bars in A and B, 1 mm; bars in C and D, 100 ��m) (TIF) Click here for additional data file.(7.8M, tif) Figure S3 Western blot analysis in cultured cells. Whole cell extracts were immunoblotted with the ind
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