RNA interference (RNAi)-mediated knockdown of target gene expression represents a powerful approach for functional genomics and therapeutic applications. However, for T lymphocytes, central regulators of immunity and immunopathologies, the application of RNAi has been limited due to the lack of efficient small interfering RNA (siRNA) delivery protocols, and an inherent inefficiency of the RNAi machinery itself. Here, we use nucleofection, an optimized electroporation approach, to deliver siRNA into primary T lymphocytes with high efficiency and negligible impairment of cell function. We identify siRNA stability within the cells as the critical parameter for efficient RNAi in primary T cells. While generally short-lived and immediately lost upon T-cell activation when conventional siRNA is used, target gene knockdown becomes insensitive to cell activation and can persist for up to 2 wk in non-dividing cells with siRNA stabilized by chemical modifications. Targeting CD4 and the transcription factor GATA-3, we show that the use of stabilized siRNA is imperative for functional gene analysis during T lymphocyte activation and differentiation in vitro as well as in vivo.Key words: Gene expression . Immune regulation . T cells Introduction RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing [1][2][3]. Given its ease of application, its high efficiency and remarkable specificity, RNAi holds great promise for broad in vitro and in vivo application in all biomedical areas. Most importantly, RNAi is directly applicable to essentially all somatic cell types including human primary cells, which makes it an invaluable tool to study human gene function and may enable new therapeutic approaches [4,5].Within the immune system T lymphocytes are one major target for siRNA-based gene silencing, since they are key regulators of immune responses and involved in many immune related disorders, like autoimmunity, chronic inflammation or lymphoma.Despite this high potential, the lack of protocols for efficient and sustained siRNA delivery into primary mammalian cells is currently the major obstacle to the use of RNAi. In particular, primary lymphocytes are highly resistant to non-viral transfection using cationic lipids and polymer reagents [6][7][8]. Although lymphocytes can be transfected by electroporation in vitro, this method so far has been rather inefficient, limited to activated cells and was complicated by a severe impairment of cell function and cell viability [9,10]. Recently, a promising approach for the 2616in vivo targeting of lymphocytes has been reported using antibody-protamine fusion proteins to deliver siRNA [11]. However, recent data from small-hairpin RNA transgenic mice indicate that in T lymphocytes the RNAi machinery itself works inefficiently as compared with other cell types [12]. In fact, due to the aforementioned problems with siRNA delivery, the parameters determining RNAi efficien...

Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis.

Several methods for the detection of RNA have been developed over time. For small RNA detection, a stem-loop reverse primerbased protocol relying on TaqMan RT-PCR has been described. This protocol requires an individual specific TaqMan probe for each target RNA and, hence, is highly cost-intensive for experiments with small sample sizes or large numbers of different samples. We describe a universal TaqMan-based probe protocol which can be used to detect any target sequence and demonstrate its applicability for the detection of endogenous as well as artificial eukaryotic and bacterial small RNAs. While the specific and the universal probe-based protocol showed the same sensitivity, the absolute sensitivity of detection was found to be more than 100-fold lower for both than previously reported. In subsequent experiments, we found previously unknown limitations intrinsic to the method affecting its feasibility in determination of mature template RISC incorporation as well as in multiplexing. Both protocols were equally specific in discriminating between correct and incorrect small RNA targets or between mature miRNA and its unprocessed RNA precursor, indicating the stem-loop RT-primer, but not the TaqMan probe, triggers target specificity. The presented universal TaqMan-based RT-PCR protocol represents a cost-efficient method for the detection of small RNAs.