Figure 3

Comprehensive and quantitative profiling of the EBV miRNA transcriptome in cells with the three latency types. (A) The comparable results from poly(A)-tailed, real-time RT-PCR analysis of the EBV-encoded miRNA transcriptome in the cell lines with latency I, II and III. The respective positions of the four clusters of EBV miRNAs (miR-BHRF1, miR-BART clusters I/II, miR-BART2 and the B95-8 deletion region) are marked below, and the numbers under the lines indicate the EBV genomic locations (nt). (B) Differential amplification curves of EBV miRNA species between epithelial and lymphoid tumor cells. The amplification curves of EBV miRNAs in C666-1 and Raji cells are separated distinctly into two parts: in epithelial C666-1 cells, the EBV BART family generally had lower Ct values than the BHRF1 family (left); and the opposite results were observed for the lymphoid Raji cells (right). (C) Verification of qPCR product specificity via DNA sequencing based on AT cloning. Representative DNA sequence results are shown for three, randomly selected qPCR products, the BHRF1 family member BHRF1-1 and two BART family members, Bart5 and Bart15. (D) An overview image from the microarray analysis of human miRNAs and EBV miRNAs in C666-1 cells (left). The right panel displays a subset of the hybridization signals for EBV miRNAs in the indicated region of the chips for tumor cell lines with various EBV latency types and including the EBV-negative cell line CNE2. (E) Correlation between the microarray read counts and the expression levels that were determined by real-time PCR of EBV miRNAs that were detected in cells with various latency types. The microarray and real-time PCR results were log2-transformed and analyzed using Person’s correlation analysis. Pearson’s correlation coefficients and p-values are shown in the inserts.