MicroRNA in Chronic Lymphocytic Leukemia Figure 2. Validation of altered miRNA expression identified by GSEA. miRNA-specific RT-PCR independently confirms altered expression of GSEA-identified miRNAs in B cells activated with CpG. A. miR-181a, miR-181b, miR-20b, miR-103, miR-15b, miR-331-3p, and let-7c. B. miR-155. C. miR- 342-3p, miR-337-3p, miR-198, and miR-34a. For (A, B, C), B (-): control B cell samples. B (+): corresponding activated B samples by CpG. Error bars indicate the standard deviation of 3 independent samples. D, E. miRNA-specific RT-PCR confirms the differential expression of subsets of GSEA- identified miRNAs in CLL relative to control B cells. Upregulated miRNAs are miR-34a, miR-155, miR-198 and miR-342-3p. Downregulated miRNA expression including let-7c, miR-15b, miR-20b, miR-103, miR-181a, miR-181b, miR-331-3p and miR-337-3p. Between 4 to 8 control unstimulated B cell samples (square) and between 6 to 21 CLL patient samples (triangle) were used. The bar indicates the average expression in each group. p-value for the analysis calculated by t-test is shown. miRNA expression was normalized by snoRNA-RNU44 expression. Relative miRNA expression was calculated by DDCt method and is indicated on the Y-axis. doi:10.1371/journal.pone.0016956.g002 two miRNAs was also associated with ZAP70+ and IgVH unmutated patients (Table S4A and S4B) implying poor prognosis. Discussion miRNA expression profiling has been used to identify signatures that classify CLL [10,11,12,33]. Interestingly, none of these published signatures are identical (Table S5). The reasons for the different miRNA signatures are unknown. Different methods and different miRNA analysis platforms may partly explain the different signatures identified by each of these groups. Calin et al. profiled 94 CLL patient samples using a miRNA microarray but did not purify CLL cells to homogeneity [10,11]. Fulci et al. profiled 56 CLL patient samples by cloning and SYBR-green- based RT-PCR [12]. Zanette et al. profiled miRNA expression in 9 CLL patients and 6 healthy donors using TaqMan-based miRNA assays [13]. However, CLL is a heterogeneous disease and it is possible that different patient populations have unique miRNA expression patterns. Interestingly, Zhang et. al. [14] demonstrated CLL cells are more like memory B cells based on miRNA profiling, our miRNA profiling data showed CLL cells are behaving like activated B cells at an earlier time point. The uniformity of miRNA expression changes in CLL patient samples relative to B cells is striking (Figure 1 and Figure S3). Though the directionality of changes in expression is consistent for these particular miRNAs, the magnitude of changes varies considerably for these particular miRNAs. Indeed, some miRNAs demonstrate expression variance over several orders of magnitude. PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e16956