living cells or after the miRNAs are released from cells into the bloodstream [64–66]. In the present study, the in-vitro transfection experiments suggest that increased expression of Ago1 and/or Ago2 genes contribute to the altered levels of miR-21 and miR-21* in the cells. The in vivo data from mouse hearts revealed age-related changes in the expression of Ago1 and Ago2 genes. However, the Ago1 and miR- 21* gene expression were decreased in 24-month-old mouse hearts vs. 4 month-old hearts; while Ago2 expression was increased in 24- month-old mouse hearts vs. 4 month-old hearts. We also observed that some miRNAs were down-regulated in the 24-month-old vs. 4-month-old hearts. It is plausible that the degree of down- regulation of Ago1 and up-regulation of Ago2, the length of the 39 UTR of the target, the relative abundance of the endogenous miRNA of interest, and the relative abundance of the target messenger RNA might influence the down-regulation of the miRNAs/clusters. 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Saito T, Saetrom P (2012) Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments. Silence 3: 3. PLoS ONE | www.plosone.org 12 April 2012 | Volume 7 | Issue 4 | e34688