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  • Cappable-seq Service now available !

    In close cooperation with New England Biolabs, Inc. (NEB), VERTIS is now able to make the new Cappable-seq method available to its customers. The method was developed by Laurence Ettwiller and Ira Schildkraut from NEB (Ettwiller, L. et al. 2016. BMC Genomics. 17, 1 (2016), 199).

    It represents the current most sensitive and robust method for the precise identification of bacterial transcription start sites (TSSs).

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WTSS FL-cDNA Normalization MicroRNA PARE DGE

cDNA Synthesis From microRNA (miRNA)

We have a strong background in the sequencing of miRNA - we were actually one of the first companies world wide, which offered professional miR sequencing services (see Berezikov et al. 2006, Nature Genetics 38: 1375-1377).

RNA in the size fraction smaller 200 nt is prepared from total RNA or from cells ore tissues using silica based small RNA isolation kits (e.g. the mirVana miRNA isolation kit from Ambion).

Smaller RNA fractions like microRNA (miRNA or miR) with a size of 15 - 30 nt are semi-automatically fractionated using the LabChip XT fractionation system from Caliper/PerkinElmer.

References

Porstner, M. et al. 2015. miR-148a promotes plasma cell differentiation and targets the germinal center transcription factors Mitf and Bach2. European Journal of Immunology. 45, 4 (2015), 1206–1215.

Chandran, A. et al. 2014. The TGF-?-inducible miR-23a cluster attenuates IFN-? levels and antigen-specific cytotoxicity in human CD8+ T cells. Journal of Leukocyte Biology. 96, 4 (2014), jlb.3A0114–025R.

Schulte, L. et al. 2011. Analysis of the host microRNA response to Salmonella uncovers the control of major cytokines by the let-7 family. The EMBO Journal. 30, 10 (2011), 1977–1989.

Schraivogel, D. et al. 2011. CAMTA1 is a novel tumour suppressor regulated by miR-9/9* in glioblastoma stem cells. The EMBO Journal. 30, 20 (2011), 4309–4322.

Brameier, M. et al. 2011. Human box C/D snoRNAs with miRNA like functions: expanding the range of regulatory RNAs. Nucleic Acids Research. 39, 2 (2011), 675–686.

Wong, C. et al. 2011. MicroRNAs in the shoot apical meristem of soybean. Journal of Experimental Botany. 62, 8 (2011), 2495–2506.

Aguilar, A. et al. 2010. The small RNA expression profile of the developing murine urinary and reproductive systems. FEBS Letters. 584, 21 (2010), 4426–4434.

Krol, J. et al. 2010. Characterizing Light-Regulated Retinal MicroRNAs Reveals Rapid Turnover as a Common Property of Neuronal MicroRNAs. Cell. 141, 4 (2010), 618–631.

Li, S. et al. 2009. Direct sequencing and expression analysis of a large number of miRNAs in Aedes aegypti and a multi-species survey of novel mosquito miRNAs. BMC Genomics. 10, 1 (2009), 581.

Zhu et al. 2009. Identification of Novel Epstein-Barr Virus MicroRNA Genes from Nasopharyngeal Carcinomas. Journal of Virology. 83, 7 (2009), 3333–3341.

De Wit, E. et al. 2009. Repertoire and evolution of miRNA genes in four divergent nematode species. Genome Research. 19, 11 (2009), 2064–2074.

Tarasov, V. et al. 2007. Differential Regulation of microRNAs by p53 Revealed by Massively Parallel Sequencing: miR-34a is a p53 Target That Induces Apoptosis and G1-arrest. Cell Cycle. 6, 13 (2007), 1586–1593.

Kloosterman, W. et al. 2006. Cloning and expression of new microRNAs from zebrafish. Nucleic Acids Research. 34, 9 (2006), 2558–2569.

Berezikov, E. et al. 2006. Diversity of microRNAs in human and chimpanzee brain. Nature Genetics. 38, 12 (2006), 1375–1377.

Berezikov et al. 2006. Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis. Genome Research. 16, 10 (2006), 1289–1298.

Example: Preparation of cDNA from microRNA

Small RNA was isolated from Arabidopsis leaves using the mirPremier microRNA Isolation kit from SIGMA. Then miR species in a size range of 15 - 30 nt were semi-automatically fractionated using the LabChip XT fractionation system from Caliper/PerkinElmer. Images of the quality controls of the different steps during miR cDNA synthesis are shown below:

 

 

The combined length of the flanking sequences (Illumina sequencing adapters and polyA tail) is about 150 bases. Therefore, PCR-products containing miR sequences of  15 - 30 nt have a total length of 165 – 180 bp.
 
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