Unknown,Transcriptomics,Genomics,Proteomics

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Identification of the 5' end of the pig miRNA by the RAKE technology

ABSTRACT: We applied a new computational approach to predict specie-specific and conserved miRNAs, than experimentally confirmed by a modified RNA-primed Array-based Klenow Extension (RAKE) method. We identified 489 conserved and 1,178 pig-specific novel miRNAs increasing our tally of confirmed miRNAs to 1,667 novel miRNAs. In addition, RAKE allowed the identification of miRNA isoforms (isomiRs) that we demonstrated to be differentially expressed across tissues suggesting that subtle variability in isomiR expression is regulated and biologically meaningful. 12K microarray was hybridized with a pool of small RNAs from 20 different tissues (superior vena cava, adipose tissue, lung, spleen, stomach, liver, intestine, kidney, descending aorta, left atrium, left ventricle, skeletal muscle, pulmonary aorta, skin, tongue, ascending aorta, arterial white cells blood, venal white cells blood, coronary valve, lymph node). Same experiment was replicated three times to grant the reliability of the identified 5M-bM-^@M-^Y end of miRNAs and microarray slides were scanned three times at low, medium and high PMT to improve microarray dynamic range.

ORGANISM(S): Sus scrofa

SUBMITTER: Gerolamo Lanfranchi

PROVIDER: E-GEOD-28139 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here we developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10-18 moles to 10-14 moles in a linear range) (see Figure), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 14 different tissues relating it with tissue anatomical proximity and functional similarity. 2K microarray was hybridized with small RNAs from 14 different tissues (Atrium Sinister; Skin; Liver; W_B_A: White Blood Cells from Arteriosum blood; W_B_V: White Blood Cells from Venosum blood; Lymph Node; Tongue; Spleen; Skeletal Muscle; Lung; Kidney; Stomach; Adipose Tissue; Ventricle Sinister). Each experiment was replicated to have 28 experiments and miRNA gene expression was correlated with mRNA gene expression in the same samples.

Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here we developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10^-18 moles to 10^-14 moles in a linear range), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 9 different tissues relating them with tissue functionality in the cardiocirculatory system.

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Identification of the 5' end of the pig miRNA by the RAKE technology

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