Concatameric cloning of porcine microRNA molecules after assembly PCR

Sharbati, Soroush; Kutz-Lohroff, Barbara; Scholven, Jutta; Einspanier, Ralf

doi:10.1016/j.bbrc.2008.08.048

Cited by 21 publications

(16 citation statements)

References 15 publications

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“…Over the course of the last three years we further established miR-Q assays for several miRNAs according to different research projects [8,17,18]. Another 21 newly established assays are presented here for the first time including a set of five snRNA specific assays serving as reference genes.…”

Section: Resultsmentioning

confidence: 99%

Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes

Sharbati

Hoeke

et al. 2012

BMC Genomics

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BackgroundSmall 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.ResultsWe 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.ConclusionsThe 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.

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Section: Resultsmentioning

confidence: 99%

Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes

Sharbati

Hoeke

et al. 2012

BMC Genomics

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“…In an earlier study, we have identified several porcine miRNAs applying a novel cloning strategy based on synthesis of concatamers [17] followed by expression profiling using a miRNA specific quantitative PCR approach [27]. Here a more straight forward approach was chosen based on new technologies to generate comprehensive data in a less time consuming manner.…”

Section: Discussionmentioning

confidence: 99%

“…However, to better investigate and understand regulatory networks of such exemplified diseases in the porcine model, a deeper coverage of the intestinal miRNAome is needed. Recently, we have reported 10 novel porcine miRNAs after introduction of a new concatameric cloning strategy [17]. Deep sequencing enables the immediate generation of a vast number of sequence data.…”

Section: Introductionmentioning

confidence: 99%

Deciphering the porcine intestinal microRNA transcriptome

et al. 2010

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BackgroundWhile more than 700 microRNAs (miRNAs) are known in human, a comparably low number has been identified in swine. Because of the close phylogenetic distance to humans, pigs serve as a suitable model for studying e.g. intestinal development or disease. Recent studies indicate that miRNAs are key regulators of intestinal development and their aberrant expression leads to intestinal malignancy.ResultsHere, we present the identification of hundreds of apparently novel miRNAs in the porcine intestine. MiRNAs were first identified by means of deep sequencing followed by miRNA precursor prediction using the miRDeep algorithm as well as searching for conserved miRNAs. Second, the porcine miRNAome along the entire intestine (duodenum, proximal and distal jejunum, ileum, ascending and transverse colon) was unraveled using customized miRNA microarrays based on the identified sequences as well as known porcine and human ones. In total, the expression of 332 intestinal miRNAs was discovered, of which 201 represented assumed novel porcine miRNAs. The identified hairpin forming precursors were in part organized in genomic clusters, and most of the precursors were located on chromosomes 3 and 1, respectively. Hierarchical clustering of the expression data revealed subsets of miRNAs that are specific to distinct parts of the intestine pointing to their impact on cellular signaling networks.ConclusionsIn this study, we have applied a straight forward approach to decipher the porcine intestinal miRNAome for the first time in mammals using a piglet model. The high number of identified novel miRNAs in the porcine intestine points out their crucial role in intestinal function as shown by pathway analysis. On the other hand, the reported miRNAs may share orthologs in other mammals such as human still to be discovered.

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“…Using small RNA cloning, SharbatiTehrani et al identified 11 miRNAs from various pig tissues (Sharbati-Tehrani et al, 2008). Combining deep sequencing and computational analysis, 777 unique miRNAs were identified from six prenatal and four postnatal pigs, including 30 tissue-specific miRNAs .…”

Section: Mirnas In Pigsmentioning

confidence: 99%

MicroRNAs in farm animals

Wang

Jiang

2013

Animal

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MicroRNAs (miRNAs) are a class of ,22 nucleotide-long small noncoding RNAs that target mRNAs for translational repression or degradation. miRNAs target mRNAs by base-pairing with the 3 0 -untranslated regions (3 0 -UTRs) of mRNAs. miRNAs are present in various species, from animals to plants. In this review, we summarize the identification, expression, and function of miRNAs in four important farm animal species: cattle, chicken, pig and sheep. In each of these species, hundreds of miRNAs have been identified through homology search, small RNA cloning and next generation sequencing. Real-time RT-PCR and microarray experiments reveal that many miRNAs are expressed in a tissue-specific or spatiotemporal-specific manner in farm animals. Limited functional studies suggest that miRNAs have important roles in muscle development and hypertrophy, adipose tissue growth, oocyte maturation and early embryonic development in farm animals. Increasing evidence suggests that single-nucleotide polymorphisms in miRNA target sites or miRNA gene promoters may contribute to variation in production or health traits in farm animals.

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Concatameric cloning of porcine microRNA molecules after assembly PCR

Cited by 21 publications

References 15 publications

Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes

Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes

Deciphering the porcine intestinal microRNA transcriptome

MicroRNAs in farm animals

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