title: Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes.
abstract: BACKGROUND
  Small interfering and non-coding RNAs regulate gene expression across all kingdoms of life. MicroRNAs constitute an important group of metazoan small RNAs regulating development but also disease. Accordingly, in functional genomics microRNA expression analysis sheds more and more light on the dynamic regulation of gene expression in various cellular processes.
  
  
  RESULTS
  We have developed custom RT-qPCR arrays allowing for accurate quantification of 31 small RNAs in triplicate using a 96 well format. In parallel, we provide accurate normalisation of microRNA expression data based on the quantification of 5 reference snRNAs. We have successfully employed such arrays to study microRNA regulation during human monocyte differentiation as well as Salmonella infection. Besides well-known protagonists such as miR-146 or miR-155, we identified the up-regulation of miR-21, miR-222, miR-23b, miR-24, miR-27a as well as miR-29 upon monocyte differentiation or infection, respectively.
  
  
  CONCLUSIONS
  The provided protocol for RT-qPCR arrays enables straight-forward microRNA expression analysis. It is fully automatable, compliant with the MIQE guidelines and can be completed in only 1 day. The application of these arrays revealed microRNAs that may mediate monocyte host defence mechanisms by regulating the TGF-β signalling upon Salmonella infection. The introduced arrays are furthermore suited for customised quantification of any class of small non-coding RNAs as exemplified by snRNAs and thus provide a versatile tool for ubiquitous applications.
authors.affiliation: Institute of Veterinary Biochemistry, Freie Universitaet Berlin, Berlin, Germany. sharbati@zedat.fu-berlin.de
authors.forename: Soroush
authors.initials: S
authors.lastname: Sharbati
authors.forename: Jutta
authors.initials: J
authors.lastname: Sharbati
authors.forename: Lena
authors.initials: L
authors.lastname: Hoeke
authors.forename: Marc
authors.initials: M
authors.lastname: Bohmer
authors.forename: Ralf
authors.initials: R
authors.lastname: Einspanier
mesh_terms: D002454:Cell Differentiation; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism* / Q000382:microbiology; D020411:Oligonucleotide Array Sequence Analysis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012475:Salmonella / Q000502:physiology*; D012480:Salmonella Infections / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D016212:Transforming Growth Factor beta