Chemical capping improves template switching and enhances sequencing of small RNAs

Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5′ cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5′ cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA—a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.

Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids

Background Extracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. Several RNA isolation methods are available for the analysis of these cell-free miRNAs by RT-qPCR. Not all methods, however, are equally suitable for different biofluids. Here, we extracted total RNA from four very diverse biofluids: serum, urine, bile, and graft preservation fluid (perfusate). Four different protocols were used: a phenol-chloroform extraction and alcohol precipitation in combination with a precipitation carrier (QP) and three different column-based isolation methods, one with phenol-chloroform extraction (RN) and two without (NG and CU). For this range of clinical biofluid samples, we evaluated the potential of these different RNA isolation methods assessing recovery efficiency and the co-purification of RT-qPCR inhibiting compounds. Results Differences were observed between each of the RNA isolation methods in the recovery of cel-miR-39, a synthetic miRNA spiked in during the workup procedure, and for endogenous miRNAs. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed using heparinase I during cDNA synthesis. RT-qPCR detection of synthetic miRNAs cel-miR-39, spiked in during RNA workup, cel-miR-54, spiked in during cDNA synthesis, and endogenous miRNAs was strongly improved in the presence of heparinase I for some, but not all, isolation methods. Other, co-isolated RT-qPCR inhibitors were not identified, except for biliverdin, which co-isolated from some bile samples with one of the methods. In addition, we observed that serum and urine contain compounds that enhance the binding of heparin to certain solid-phase columns. Conclusions For reliable measurements of miRNA-based biomarkers in biofluids, optimization of RNA isolation procedures is recommended as methods can differ in miRNA detection and in co-purification of RT-qPCR inhibitory compounds. Heparinase I treatment confirmed that heparin appeared to be the major RT-qPCR inhibiting compound, but also biliverdin, co-isolated from bile, could interfere with detection.

Sensing single molecule under micromanipulation to quantify nucleic acids

Rapid molecular diagnosis using nucleic acid biomarkers is important for timely identification of acute pathogenic infections. We introduce an active kinetic approach called sensing single molecule under micromanipulation (SSM3) to quantify nucleic acids, which circumvents hydrodynamic limits of reaction rate, sensitivity and specificity. We demonstrate the 15-minute assay to detect synthetic miRNAs with subfemtomolar limit of detection and high-confidence discrimination of single-base-pair mismatch.

Serum Derived Extracellular Vesicles Mediated Delivery of Synthetic miRNAs in Human Endothelial Cells

Extracellular vesicles (EVs) have emerged in the last decades as a cell-to-cell communication mechanism. One of their mechanism of action is the direct delivery of their cargo, composed of bioactive molecules to target cells. Different methods (direct electroporation, cell transfection, chemical transfection) were developed to vehicle therapeutic molecules through EVs. However, most of these techniques presented some limitations such as EV disruption and aggregation. In the present study, we demonstrated that a direct temperature-controlled co-incubation of EVs with defined miRNAs is a stable method to deliver information to target cells without affecting EV constitutive content. We chose serum as an easy and abundant source of EVs applicable to autologous treatment after EV modification. Exogenous cel-miR-39 loaded on serum EVs (SEVs) was taken up by human endothelial cells, demonstrating an adequate miRNA loading efficacy based on the co-incubation method. Moreover, SEVs co-incubation with the angiomiRNA-126 (miR-126) enhanced their angiogenic properties in vitro and in vivo by increasing the capacity to induce capillary-like structure formation of human endothelial cells. MiR-126 loaded EVs were also shown to stimulate mouse endothelial cells to invade Matrigel plugs and create more vessels with respect to the EV naive counterpart. When SEVs were loaded with miR-19b, an anti-angiogenic miRNA, they were able to reduce Vascular endothelial growth factors (VEGF) pro-angiogenic capacity, supporting the selective biological effect mediated by the carried miRNA. Lastly, we identified Annexin A2 (ANXA2) as one of the molecules involved in the exogenous RNA binding to serum EV surface, favoring miRNA delivery to target endothelial cells for potential therapeutic application.

Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids.

BACKGROUND Extracellular microRNAs (miRNAs), released from cells into biofluids, have emerged as promising biomarkers for diagnostic and prognostic purposes. For the analysis of these cell-free miRNAs in various biofluids by RT-qPCR, several RNA isolation methods are available. However, not all methods are equally suitable for different biofluids. The aim of this study is to evaluate the potential of different RNA isolation methods in a range of clinical biofluid samples.METHODS Total RNA was isolated from serum, urine, bile, and graft preservation fluid (perfusate) using four different protocols: phenol-chloroform extraction in combination with a precipitation carrier, and three different column-based isolation methods. Co-purification of heparin, a known RT-qPCR inhibitor, was assessed using heparinase I during cDNA synthesis. Synthetic miRNAs, spiked-in during RNA workup (cel-miR-39) or during cDNA synthesis (cel-miR-54), and endogenous miRNAs were quantified using RT-qPCR.RESULTS Recovery of cel-miR-39 significantly differed between methods with miRNeasy columns providing the best overall recovery in the four biofluids tested, as was also observed for endogenous miRNAs. Contamination of RNA with heparin differed between sample type and isolation method, and could be counteracted using heparinase I. Other co-isolated RT-qPCR inhibitors were not identified, except for biliverdin which co-isolated from some bile samples with one of the methods.CONCLUSIONS For reliable measurements of miRNA-based biomarkers in biofluids, optimization of RNA isolation procedures is recommended as methods can differ in miRNA detection and co-purification of RT-qPCR inhibitory compounds. Heparinase I treatment confirmed that heparin appeared to be the major RT-qPCR-inhibiting compound but also biliverdin, co-isolated from bile, could interfere with detection.

Development of MicroRNAs as Potential Therapeutics against Cancer

MicroRNAs (miRNAs) are small noncoding RNAs that function at the posttranscriptional level in the cellular regulation process. miRNA expression exerts vital effects on cell growth such as cell proliferation and survival. In cancers, miRNAs have been shown to initiate carcinogenesis, where overexpression of oncogenic miRNAs (oncomiRs) or reduced expression of tumor suppressor miRNAs has been reported. In this review, we discuss the involvement of miRNAs in tumorigenesis, the role of synthetic miRNAs as either mimics or antagomirs to overcome cancer growth, miRNA delivery, and approaches to enhance their therapeutic potentials.

An extension to: Systematic assessment of commercially available low-input miRNA library preparation kits

High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. To assist researchers choose the most appropriate library preparation kit, we recently compared the performance of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. As before, performance was assessed with respect to sensitivity, reliability, titration response and differential expression. Despite NEXTflex employing partially-randomised adapter sequences to minimise bias, we reaffirm that biases in miRNA abundance are kit-specific, complicating the comparison of miRNA datasets generated using different kits.

Comparison of miRNA profiling methods using synthetic miRNA pools and standardized exRNA samples reveals substantial performance differences

SUMMARYMicroRNAs (miRNAs) found in biofluids play functional roles in health and in disease pathogenesis, underpinning their potential as clinical biomarkers. Several platforms for measurement of extracellular RNAs have recently become available. We evaluated the reproducibility, accuracy, sensitivity, and specificity of four miRNA quantification platforms, including one widely used discovery approach (small RNA-seq) and three targeted platforms (FirePlex, EdgeSeq, and nCounter). Using pools of synthetic miRNAs, we observed that reproducibility was highest for RNA-seq and EdgeSeq, that all three targeted platforms had lower bias than RNA-seq, and that RNA-seq had the best ability to distinguish between present and absent sequences. Overall reproducibility was lower for plasma samples than synthetic miRNA pools. We compared expression of placental miRNAs in plasma from pregnant and non-pregnant women and observed expected differences with RNA-seq and EdgeSeq, but not FirePlex or nCounter. We conclude that differences in performance among miRNA profiling platforms impact their relative utility as potential assay systems for clinical biomarkers.

Engineered resistance to Zika virus in transgenic Ae. aegypti expressing a polycistronic cluster of synthetic miRNAs

Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with Wolbachia to reduce population size and/or vectoring competency, and gene drive based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered microRNAs (miRNAs) targeting ZIKV is expressed and fully processed following a blood meal in Ae. aegypti, ensuring the formation of mature synthetic miRNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered Ae. aegypti mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses.One Sentence SummaryHere we describe the generation of Ae. aegypti mosquitoes that are engineered to confer reduced vector competence to Zika virus (ZIKV) and we discuss how such engineering approach can be used to combat the major health burden of ZIKV and potentially other arboviruses in the future.