Absolute and relative quantification of placental specific microRNAs in maternal circulation in placental insufficiency related complications

Ilona Hromadnikova1, Katerina Kotlabova1, Jindrich Doucha2, Klara Dlouha3
1Third Faculty of Medicine, Charles University Prague, Czech Republic; 2Clinic of Obstetrics and Gynecology, University Hospital Motol, Prague, Czech Republic; 3Institute for the Care of the Mother and Child, Prague, Czech Republic

Abstract
The primary goal of our study has been to identify placental specific microRNAs present in maternal plasma differentiating between normal pregnancies and non-pregnant individuals. The selection of appropriate pregnancy associated microRNAs with the diagnostical potential was based on following criteria: (1) detection rate of 100 % in full-term placentas, (2) detection rate of above 67 % in maternal plasma throughout gestation (at least 4 positive wells out of 6 tested wells) and (3) detection rate of 0 % in whole peripheral blood and plasma samples of non-pregnant individuals. Initially, we tested microRNAs (miR-34c, miR-372, miR-135b and miR-518b) which had been previously identified as pregnancy-associated miRNAs. Additionally we selected 16 other highly specific placental miRNAs (miR-512-5p, miR-515-5p, miR-224, miR-516-5p, miR-517*, miR-136, miR-518f*, miR-519a, miR-519d, miR-519e, miR-520a*, miR-520h, miR-524-5p, miR-525, miR-526a and miR-526b) from the miRNAMap database. Seven microRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) were newly identified as pregnancy associated with diagnostic potential. Further, we examined if extracellular placental specific microRNAs (miR-520a*, miR-520h, miR-525 and miR-526a) can differentiate pregnancies with placental insufficiency related complications from normal ones. Absolute and relative quantification of placental specific microRNAs (miR-520a*, miR-520h, miR-525 and miR-526a) was determined in 50 normal pregnancies, 32 complicated pregnancies (21 preeclampsia with or without intrauterine growth retardation and 11 IUGR) and 3 pregnancies at various gestational stages who later developed preeclampsia w/o IUGR and/or IUGR using real-time PCR and the comparative Ct method relative to ubiquitous miR-16. Both quantification approaches revealed significant increase of extracellular placental specific microRNAs levels over time in normally progressing pregnancies, however did not differentiate between normal and complicated pregnancies at the time of preeclampsia and/or IUGR onset. Nevertheless, significant elevation of extracellular microRNAs was observed during early gestation (within 12th to 16th weeks) in pregnancies with later onset of preeclampsia and/or IUGR. To avoid instability of extracellular microRNAs in maternal plasma, prenatal monitoring should be performed using only the samples with the storage life below two months. Early gestation extracellular microRNAs screening may differentiate between normal pregnancies and those who will later develop placental insufficiency related complications.
Acknowledgements: This work was supported by grant projects MSM 0021620806 and GAUK 260707/SVV/2010.

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RNAi and gene expression profiling as a tool in cancer research

Michelle Plusquin1, An-Sofie Stevens1, Katrien De Mulder2, Frank Vanbelleghem1, Peter Ladurner2, Ann Cuypers1, Tom Artois1, Karen Smeets1
1Hasselt University, Belgium; 2University of Innsbruck

Abstract
As innovative model organisms in stem cell research, the flatworm species Schmidtea mediterranea and Macrostomum lignano were used to characterize carcinogenic events. To discover potential biomarkers, RNAi knockdown and qPCR profiling were combined. Both cancer inducers as well as blockers were used and differential expression was compared. Underlying mechanisms involve oxidative stress related processes resulting in altered gene expression profiles in function of (stem) cell proliferation. As heat shock proteins (HSP) appear to play a significant role herein, the effect of a chemotherapeutic HSP blocker was evaluated. The value of both RNAi experiments as well as gene expression profiling is highlighted in this presentation, and an overview of carcinogenic stress mechanisms and their interactions is given.

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Impact of different normalization strategies on miRNA profiling experiments

Swanhild Meyer1, Sebastian Kaiser2, Carola Wagner3, Christian Thirion4, Michael Pfaffl1
1Physiology Weihenstephan, Technische Universität München, Germany; 2Department of Statistics, Ludwig-Maximilians-Universität München, Germany; 3IMGM Laboratories GmbH, Martinsried, Germany; 4Friedrich-Baur-Institute and Department of Neurology, Ludwig-Maximilians-Universität München, Germany

Abstract
Selection of the normalization strategy has significant impact on the detection of differentially expressed microRNAs (miRNAs) from profiling experiments. Normalization techniques currently in use for miRNA profiling analyses are in analogy to mRNA data processing or are specifically modified and developed for miRNA data. Studies evaluating the impact of normalization on miRNA profiling experiments have focused on different profiling platforms or the comparison of normalization techniques within one platform. Here, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of a one-colour hybridization-based platform (AGL array) and a multiplex RT-qPCR platform (TLDA) and validated results by singleplex RT-qPCR assays.

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MicroRNA Target Validation with MISSION® 3’UTR Lenti GoClone™ and Human MicroRNA Mimics

Nikos Hontzeas
Sigma Aldrich, United States of America

Abstract
MicroRNAs (miRNAs) may regulate several hundreds of genes to control a cell’s response to developmental and environmental signals. The validation of potential target genes is essential in determining a miRNA’s role and function in these pathways. Here we use MISSION® 3′UTR Lenti GoClone™ and Human MicroRNA Mimics to demonstrate validation of known and conserved miRNA targets. Known targets of hsa-miR-29b (MCL1), hsa-miR-124 (MAPK14), and hsa-miR-373 (LATS2), and targets of conserved miRNAs hsa-miR-373 (RBL2) and hsa-miR-10a, (HOXD10), were down-regulated by their respective mimic. These result demonstrate that the use of MISSION® 3′UTR Lenti GoClone™ with Human MicroRNA Mimics is a viable option for miRNA target validation.

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Towards blood-based cancer screening using LNA™-enhanced microRNA qPCR

Ditte Andreasen, Niels Tolstrup, Jacob Ulrik Fogh, Søren Jensby Nielsen, Kim Bundvig Barken, Adam Baker, Peter Mouritzen
Exiqon, Denmark

Abstract
microRNAs (miRNAs) constitute a recently discovered class of small RNAs (typically 21-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, and miRNAs have been proposed to be master regulators of cellular state. Indeed, changes in miRNA expression patterns have been associated with disease states, including a diverse array of human cancers. Furthermore, the high stability of miRNA in common clinical source materials (e.g. FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. To facilitate discovery and clinical transfer of miRNA-based diagnostic markers, we developed the miRNome-wide LNA™-based miRCURY LNA™ Universal RT microRNA PCR platform with unparalleled sensitivity and robustness. Using a specialized design algorithm (available as a web-tool for custom design of RNA species < 30 nt), we have designed assays for human and rodent miRNAs. The platform uses only 40 ng total RNA in just a single RT reaction to profile >700 human miRNAs in two predefined 384 well plates and thus allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Following screening, it is possible to choose a sub-set of miRNA assays for validation to be formatted in 96- or 384-well plates using our Pick&Mix platform. The high sensitivity of the assays makes it possible to do high quality microRNA expression profiling in samples that contain very little total RNA, such as sections of formalin fixed paraffin-embedded samples (FFPE) and blood serum and plasma. Results will be presented demonstrating the application of the new qPCR method in the profiling of miRNAs from plasma as part of our efforts to develop molecular diagnostic tests for treatment selection, diagnosis and monitoring of cancer. We will also show results demonstrating how selection of a proper set of miRNA biomarkers in a Pick&Mix plate format can give precise classification of disease progression.

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Development of an ultra-high throughput long non-coding RNA qPCR screening system

Jan Hellemans1, Pieter Mestdagh2, Steve Lefever2, Barbara D’haene1, Filip Pattyn2, Frank Speleman2, Jo Vandesompele1
1Biogazelle, Ghent, Belgium; 2Ghent University, Ghent, Belgium

Abstract
Long non-coding RNAs (lncRNA) are an underexplored class of non-coding RNAs and have been shown to be implicated in health and disease. They constitute a new class of biomarkers with disease associated lncRNA signatures yet to be discovered. In addition, they open up a new approach to understand the function and organization of the genome. The lack of a high-throughput platform to detect and quantify lncRNAs has hampered their study so far. To address this, we developed a new platform for ultra-high throughput RT-qPCR analysis of long non-coding RNAs. In the pilot phase, we designed qPCR assays for +1000 lncRNAs based on public sequence databases and lincRNA chromatin signatures. All assays were designed using state-of-the-art in silico quality controls, followed by extensive empirical validation according to MIQE guidelines. Validation of this first set in relevant biological model systems demonstrated excellent sensitivity and specificity of the technology. Data processing was done using the qbasePLUS software with an improved global mean normalization procedure to better remove technical variation. In conclusion, we have successfully completed the first stage of the development process of an ultra-high throughput and low-volume RT-qPCR platform for the quantitative detection of lncRNAs. The tool offers a unique way to investigate the expression patterns of lncRNAs in health and disease.

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