Normal miRNA biogenesis and effective knockdown of A1 mRNA in transgenic mice. (A) To assess the impact of transgene expression on endogenous miRNA levels of miR16 and miR142b in spleens from WT, VVA1.2, and DT mice were assessed by N-Code PCR analysis on reverse-transcribed miRNAs. U6 ribosomal RNA was amplified for reference. (B-C) siRNAs targeting firefly luciferase or A1 were assessed by N-Code PCR analysis on reverse-transcribed miRNA samples extracted from the spleen of mice of the indicated genotypes and in spleen cells derived from DT mice after 32 days of doxycycline application in the drinking water. (D) cDNA was generated from splenocytes of WT mice, sorted eGFP⁺⁺ cells from untreated DT mice or DT mice kept on doxycycline for 32 days. Knockdown efficiency was evaluated by quantitative RT-PCR on cDNA using primers amplifying all A1 isoforms yielding a 743-bp product (top panel). PCR products were purified and digested by restriction enzymes BglII and NsiI to confirm knockdown of all major A1 isoforms present in spleen (see also supplemental Figure 1 for further details). Images have been gray-scale inverted for easier visualization of cleaved isoforms. (E) cDNA was generated from total spleen of WT or VVA1.2 mice as well as from FACS-sorted Venus⁺ or Venus⁻ spleen cells from VVA1.1 mice. Knockdown efficiency was evaluated by quantitative RT-PCR on cDNA using primers amplifying all A1 isoforms or HPRT for normalization. (F) cDNA was generated from splenocytes of WT, TREA1 single-transgenic, or DT mice, expressing different levels of eGFP. Knockdown efficiency was evaluated as in panel D. Results of 3 independent experiments and cDNA samples from 5 animals/genotype are represented as box plots. Box length equals interquartile range. Circles represent minimal and maximal values.