Fachbereich Veterinärmedizin



    miR-Q: a novel quantitative RT-PCR approach for expression profiling of small RNA molecules like miRNAs in a complex sample (2007)

    Sharbati-Tehrani, S.
    Kutz-Lohroff, B.
    Bergbauer, R.
    Scholven, J.
    Einspanier, R.
    GBM Fall Meeting - Molecular Life Sciences 2007
    Hamburg, 16. – 19.09.2007
    URL (Volltext): http://www.doi.org
    DOI: 10.1240/sav_gbm_2007_h_001831
    Institut für Veterinär-Biochemie

    Oertzenweg 19 b
    14163 Berlin
    Tel.+49 30 838 62225 Fax.+49 30 838-62584

    Abstract / Zusammenfassung

    MicroRNAs (miRNAs) are regarded as major regulators in eukaryotic gene expression. MiRNAs represent highly conserved families of single-stranded small noncoding RNA molecules of approximately 22 nt in length. Northern Blot analysis and Microarrays are employed for miRNA profiling. An alternative and highly reliable method for accurate differential gene expression analysis is considered to be quantitative reverse transcription PCR (qRT-PCR). Currently, few miRNA qRT-PCR approaches are introduced, which are based on TaqMan assays (Chen et al., 2005) or SYBR green chemistry using LNA-modified primers (Raymond et al., 2005).
    Here we present the miR-Q assay, a new cost-effective and highly sensitive method neither requiring the use of fluorochromic probes nor LNA-modified oligonucleotides. First, miRNAs are converted into cDNA by reverse transcription using a miRNA-specific oligonucleotide with 5? overhang (RT6-miR-x). Afterwards, cDNA-molecules are quantified by a novel PCR approach based on utilising three DNA-oligonucleotides at different concentrations. A first oligonucleotide (short-miR-x-rev) comprises a sequence homology to the miRNA molecule with an additional 5? overhang. The introduced 5? overhangs provide binding sequences for universal primers (MP-fw & MP-rev), which prime the final quantitative PCR reaction utilising the SYBR green chemistry. A major advantage is that universal primers can be employed for diverse miRNAs. This approach provides a remarkable increase of specificity and simplified detection of small RNAs. It shows a high dynamic range of 6 to 8 orders of magnitude comprising a sensitivity of up to 0.2 fM synthetic miRNA per miR-Q reaction. Using this approach, we quantified the expression of miR-145 and miR-21 in different porcine intestinal samples (European Patent Application No. 07006897.8).