miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are small endogenous non-coding interfering RNA molecules regarded as major regulators in eukaryotic gene expression. Different methods are employed for miRNA expression profiling. For a better understanding of their role in essential biological processes, convenient methods for differential miRNA expression analysis are required.</p> <p>Results</p> <p>Here, we present the miR-Q assay as a highly sensitive quantitative reverse transcription PCR (qRT-PCR) for expression analysis of small RNAs such as miRNA molecules. It shows a high dynamic range of 6 to 8 orders of magnitude comprising a sensitivity of up to 0.2 fM miRNA, which corresponds to single copies per cell. There is nearly no cross reaction among closely-related miRNA family members, which points to the high specificity of the assays. Using this approach, we quantified the expression of let-7b in different human cell lines as well as miR-145 and miR-21 expression in porcine intestinal samples.</p> <p>Conclusion</p> <p>miR-Q is a cost-effective and highly specific approach, which neither requires the use of fluorochromic probes, nor Locked Nucleic Acid (LNA)-modified oligonucleotides. Moreover, it provides a remarkable increase in specificity and simplified detection of small RNAs.</p

Integrated MicroRNA-mRNA-Analysis of Human Monocyte Derived Macrophages upon Mycobacterium avium subsp. hominissuis Infection

Many efforts have been made to understand basal mechanisms of mycobacterial infections. Macrophages are the first line of host immune defence to encounter and eradicate mycobacteria. Pathogenic species have evolved different mechanisms to evade host response, e.g. by influencing macrophage apoptotic pathways. However, the underlying molecular regulation is not fully understood. A new layer of eukaryotic regulation of gene expression is constituted by microRNAs. Therefore, we present a comprehensive study for identification of these key regulators and their targets in the context of host macrophage response to mycobacterial infections.We performed microRNA as well as mRNA expression analysis of human monocyte derived macrophages infected with several Mycobacterium avium hominissuis strains by means of microarrays as well as quantitative reverse transcription PCR (qRT-PCR). The data revealed the ability of all strains to inhibit apoptosis by transcriptional regulation of BCL2 family members. Accordingly, at 48 h after infection macrophages infected with all M. avium strains showed significantly decreased caspase 3 and 7 activities compared to the controls. Expression of let-7e, miR-29a and miR-886-5p were increased in response to mycobacterial infection at 48 h. The integrated analysis of microRNA and mRNA expression as well as target prediction pointed out regulative networks identifying caspase 3 and 7 as potential targets of let-7e and miR-29a, respectively. Consecutive reporter assays verified the regulation of caspase 3 and 7 by these microRNAs.We show for the first time that mycobacterial infection of human macrophages causes a specific microRNA response. We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs. This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response. In addition, functional relevance was confirmed by uncovering the control of major caspases 3 and 7 by let-7e and miR-29a, respectively

MiR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample-0

Ang () and six complementary bases (red). Detection and amplification of the relating cDNA are employed, using a novel PCR approach with three different oligonucleotides at different concentrations within the same assay. The cDNA sequence (blue) is first detected and elongated by a specific oligonucleotide with 5' overhang (). Exponential amplification is then performed using two terminal universal primers (&).<p><b>Copyright information:</b></p><p>Taken from "miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample"</p><p>http://www.biomedcentral.com/1471-2199/9/34</p><p>BMC Molecular Biology 2008;9():34-34.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2374797.</p><p></p

MiR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample-2

Ising a sensitivity of up to 0.2 fM synthetic miRNA. A) Dynamic ranges of miR-Q assays: miR-200c (black triangles), miR-145 (black circles), and miR-27a (black rhombi). B) Dynamic ranges of miR-Q assays: miR-21 (red rhombi), miR-16 (red squares), miR-23b (red triangles), and miR-141 (red circles). C) Dynamic ranges of miR-Q assays: let-7a (blue triangles), let-7b (blue rhombi), and let-7c (blue circles).<p><b>Copyright information:</b></p><p>Taken from "miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample"</p><p>http://www.biomedcentral.com/1471-2199/9/34</p><p>BMC Molecular Biology 2008;9():34-34.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2374797.</p><p></p

MiR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample-6

Ang () and six complementary bases (red). Detection and amplification of the relating cDNA are employed, using a novel PCR approach with three different oligonucleotides at different concentrations within the same assay. The cDNA sequence (blue) is first detected and elongated by a specific oligonucleotide with 5' overhang (). Exponential amplification is then performed using two terminal universal primers (&).<p><b>Copyright information:</b></p><p>Taken from "miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample"</p><p>http://www.biomedcentral.com/1471-2199/9/34</p><p>BMC Molecular Biology 2008;9():34-34.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2374797.</p><p></p

MiR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample-4

Mples (50 ng/μl) were either spiked with 100 pM synthetic let-7b (A549 si, HeLa si, and HT-29 si) or remained non-spiked (A549, HeLa, and HT-29). RT reactions were performed with 50 ng, 25 ng, and 5 ng of all RNA samples, followed by qPCR detection of let-7b in different runs. Columns represent the mean (± SD) of three measurements. A) let-7b quantification by means of the miR-Q approach using both the spike-in controls and the non-spiked samples. B) Quantification of let-7b by means of mirVana™ qRT-PCR using both the spike-in controls and the non-spiked samples.<p><b>Copyright information:</b></p><p>Taken from "miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample"</p><p>http://www.biomedcentral.com/1471-2199/9/34</p><p>BMC Molecular Biology 2008;9():34-34.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2374797.</p><p></p

MiR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample-5

Presents the mean (± SD) of three measurements. A) Expression of miR-145 in ileal samples from ten 31-day-old piglets. B) Expression of miR-145 in jejunal samples from ten 31-day-old piglets. C) Expression of miR-21 in ileal samples from ten 31-day-old piglets. D) Expression of miR-21 in jejunal samples from ten 31-day-old piglets.<p><b>Copyright information:</b></p><p>Taken from "miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample"</p><p>http://www.biomedcentral.com/1471-2199/9/34</p><p>BMC Molecular Biology 2008;9():34-34.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2374797.</p><p></p

MiR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample-3

He miR-Q assay turned out to be 2 fM synthetic let-7b (white columns). Each column represents the mean (± SD) of three measurements.<p><b>Copyright information:</b></p><p>Taken from "miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample"</p><p>http://www.biomedcentral.com/1471-2199/9/34</p><p>BMC Molecular Biology 2008;9():34-34.</p><p>Published online 10 Apr 2008</p><p>PMCID:PMC2374797.</p><p></p