Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

Use of the QIAGEN GeneReader NGS system for detection of KRAS mutations, validated by the QIAGEN Therascreen PCR kit and alternative NGS platform.

BACKGROUND: The detection of somatic mutations in primary tumors is critical for the understanding of cancer evolution and targeting therapy. Multiple technologies have been developed to enable the detection of such mutations. Next generation sequencing (NGS) is a new platform that is gradually becoming the technology of choice for genotyping cancer samples, owing to its ability to simultaneously interrogate many genomic loci at massively high efficiency and increasingly lower cost. However, multiple barriers still exist for its broader adoption in clinical research practice, such as fragmented workflow and complex bioinformatics analysis and interpretation. METHODS: We performed validation of the QIAGEN GeneReader NGS System using the QIAact Actionable Insights Tumor Panel, focusing on clinically meaningful mutations by using DNA extracted from formalin-fixed paraffin-embedded (FFPE) colorectal tissue with known KRAS mutations. The performance of the GeneReader was evaluated and compared to data generated from alternative technologies (PCR and pyrosequencing) as well as an alternative NGS platform. The results were further confirmed with Sanger sequencing. RESULTS: The data generated from the GeneReader achieved 100% concordance with reference technologies. Furthermore, the GeneReader workflow provides a truly integrated workflow, eliminating artifacts resulting from routine sample preparation; and providing up-to-date interpretation of test results. CONCLUSION: The GeneReader NGS system offers an effective and efficient method to identify somatic (KRAS) cancer mutations.

Integrated expression profiling of multiple RNA species by real-time PCR.

MicroRNAs (miRNAs) are endogenous, non-coding RNAs comprising approximately 21-23 nucleotides that regulate gene expression by binding to and targeting messenger RNA (mRNA) for translational repression or degradation. miRNAs have been shown to regulate cellular processes including proliferation, differentiation, and development and to play an important role in immune system function. The expression of miRNAs is misregulated in numerous diseases, including cancers of immunological origin. To better understand the role of miRNA in T-cell activation, we used a real-time PCR-based system to analyze changes in miRNA expression following activation of Jurkat T-cells with the inducing agents Phorbol Myristyl Acetate (PMA) and Ionomycin (CI) and detected several miRNAs that showed differential regulation following treatment. Using this system, miRNAs and their mRNA targets, along with other non-coding RNAs, can be simultaneously detected and quantified using SYBR® Green real time-PCR, enabling comprehensive, genome-wide expression profiles of multiple RNA species.

The role of upregulated miRNAs and the identification of novel mRNA targets in prostatospheres.

TICs are characterized by their ability to self-renew, differentiate and initiate tumor formation. miRNAs are small noncoding RNAs that bind to mRNAs resulting in regulation of gene expression and biological functions. The role of miRNAs and TICs in cancer progression led us to hypothesize that miRNAs may regulate genes involved in TIC maintenance. Using whole genome miRNA and mRNA expression profiling of TICs from primary prostate cancer cells, we identified a set of up-regulated miRNAs and a set of genes down-regulated in PSs. Inhibition of these miRNAs results in a decrease of prostatosphere formation and an increase in target gene expression. This study uses genome-wide miRNA profiling to analyze expression in TICs. We connect aberrant miRNA expression and deregulated gene expression in TICs. These findings can contribute to a better understanding of the molecular mechanisms governing TIC development/maintenance and the role that miRNAs have in the fundamental biology of TICs.

Optimized Workflow for Whole Genome and Transcriptome Next-Generation Sequencing of Single Cells or Limited Nucleic Acid Samples.

Whole genome and whole transcriptome sequencing require orders of magnitude more of starting nucleic acid than what is found in single cells or other extremely limited samples. High fidelity amplification of this minute amount of nucleic acids is essential to overcome the limitations caused by the low input, degradation and contamination, and to ensure a sufficient amount of DNA for preparation of high complex and high quality next-generation sequencing (NGS) libraries. Recent technical advances in multiple displacement amplification (MDA) enable studies of rare cell types, heterogeneity of body fluids, tissues, environmental samples, and organisms that cannot be cultured. Several strategies for amplification of limiting amounts of nucleic acid have been described, with PCR being popular. However, PCR-based methods result in high error rates, lower library complexity, and lower coverage uniformity. In this article, a HiFi MDA is used to accurately amplify the limited material and to allow library preparation starting from high input, while reducing PCR cycling to achieve sufficient library yields. This article describes a complete workflow from cells and small quantities of DNA or RNA to NGS libraries for Illumina sequencing instruments. © 2023 QIAGEN GmbH. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Whole genome amplification from single cells Support Protocol 1: PicoGreen™ quantification of MDA amplified DNA Support Protocol 2: Purification of amplified DNA after MDA Basic Protocol 2: Whole transcriptome amplification from single cells Alternate Protocol: Whole transcriptome amplification from purified RNA Basic Protocol 3: Enrichment of complete small genomes using target-specific primers in MDA Basic Protocol 4: Complete viral RNA amplification using target-specific primers in MDA Basic Protocol 5: Enzymatic fragmentation and adapter ligation of MDA amplified material Basic Protocol 6: Normalization of library concentration using magnetic beads.

Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions.

BACKGROUND: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. RESULTS: All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. CONCLUSION: This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.

Multiplexing siRNAs to compress RNAi-based screen size in human cells.

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study ( approximately 800 siRNAs, approximately 400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.

Concise gene signature for point-of-care classification of tuberculosis.

There is an urgent need for new tools to combat the ongoing tuberculosis (TB) pandemic. Gene expression profiles based on blood signatures have proved useful in identifying genes that enable classification of TB patients, but have thus far been complex. Using real-time PCR analysis, we evaluated the expression profiles from a large panel of genes in TB patients and healthy individuals in an Indian cohort. Classification models were built and validated for their capacity to discriminate samples from TB patients and controls within this cohort and on external independent gene expression datasets. A combination of only four genes distinguished TB patients from healthy individuals in both cross-validations and on separate validation datasets with very high accuracy. An external validation on two distinct cohorts using a real-time PCR setting confirmed the predictive power of this 4-gene tool reaching sensitivity scores of 88% with a specificity of around 75%. Moreover, this gene signature demonstrated good classification power in HIV(+) populations and also between TB and several other pulmonary diseases. Here we present proof of concept that our 4-gene signature and the top classifier genes from our models provide excellent candidates for the development of molecular point-of-care TB diagnosis in endemic areas.

Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method.

Exosomes and other extracellular vesicles (commonly referred to as EVs) have generated a lot of attention for their potential applications in both diagnostics and therapeutics. The contents of these vesicles are the subject of intense research, and the relatively recent discovery of RNA inside EVs has raised interest in the biological function of these RNAs as well as their potential as biomarkers for cancer and other diseases. Traditional ultracentrifugation-based protocols to isolate EVs are labor-intensive and subject to significant variability. Various attempts to develop methods with robust, reproducible performance have not yet been completely successful. Here, we report the development and characterization of a spin column-based method for the isolation of total RNA from EVs in serum and plasma. This method isolates highly pure RNA of equal or higher quantity compared to ultracentrifugation, with high specificity for vesicular over non-vesicular RNA. The spin columns have a capacity to handle up to 4 mL sample volume, enabling detection of low-abundance transcripts in serum and plasma. We conclude that the method is an improvement over traditional methods in providing a faster, more standardized way to achieve reliable high quality RNA preparations from EVs in biofluids such as serum and plasma. The first kit utilizing this new method has recently been made available by Qiagen as "exoRNeasy Serum/Plasma Maxi Kit".

Prophylactic and therapeutic effects of small interfering RNA targeting SARS-coronavirus.

OBJECTIVES: To identify and characterize the siRNA duplexes that are effective for inhibition of SARS-CoV infection and replication in the non-human primate cells. This in vitro study will serve as the foundation for development of novel anti-SARS therapeutics. METHODS: 48 siRNA sequences were designed for targeting regions throughout entire SARS-CoV genome RNA including open-reading frames for several key proteins. Chemically synthesized siRNA duplexes were transfected into foetal rhesus kidney (FRhK-4) cells prior to or after SARS-CoV infection. The inhibitory effects of the siRNAs were evaluated for reductions of intracellular viral genome copy number and viral titres in the cell culture medium measured by Q-RT-PCR and CPE-based titration, respectively. Four siRNA duplexes were found to achieve potent inhibition of SARS-CoV infection and replication. A prolonged prophylactic effect of siRNA duplexes with up to 90% inhibition that lasted for at least 72 h was observed. Combination of active siRNA duplexes targeting different regions of the viral genome resulted in therapeutic activity of up to 80% inhibition. CONCLUSION: Chemically synthesized siRNA duplexes targeting SARS-CoV genomic RNA are potent agents for inhibition of the viral infection and replication. The location effects of siRNAs were revealed at both genome sequence and open-reading frame levels. The rapid development of siRNA-based SARS-CoV inhibitors marked a novel approach for combating newly emergent infectious diseases.

Epidermal growth-factor-induced transcript isoform variation drives mammary cell migration.

Signal-induced transcript isoform variation (TIV) includes alternative promoter usage as well as alternative splicing and alternative polyadenylation of mRNA. To assess the phenotypic relevance of signal-induced TIV, we employed exon arrays and breast epithelial cells, which migrate in response to the epidermal growth factor (EGF). We show that EGF rapidly--within one hour--induces widespread TIV in a significant fraction of the transcriptome. Importantly, TIV characterizes many genes that display no differential expression upon stimulus. In addition, similar EGF-dependent changes are shared by a panel of mammary cell lines. A functional screen, which utilized isoform-specific siRNA oligonucleotides, indicated that several isoforms play essential, non-redundant roles in EGF-induced mammary cell migration. Taken together, our findings highlight the importance of TIV in the rapid evolvement of a phenotypic response to extracellular signals.

Analysis of putative miRNA binding sites and mRNA 3' ends as targets for siRNA-mediated gene knockdown.

Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that generate highly functional siRNAs for gene knockdown. Nevertheless, "difficult-to-silence" target messenger RNAs (mRNAs) still exist for which no functionally validated siRNAs are available. MicroRNA (miRNA) sites in the mRNA 3'UTR, which interact with miRNA-loaded RNA-induced silencing complex (miRISC) for posttranscriptional gene regulation, provide alternative potentially accessible sites for siRNA. To investigate this, we designed siRNAs directed against single putative miRNA sites (misiRNAs) as predicted by miRNA target databases as well as siRNAs against other regions within the 3'UTR of "difficult-to-silence" targets in this proof-of-principle study. Although their design was not fully optimized, the misiRNAs generally caused higher knockdown than previously designed siRNAs for these targets. In general, knockdown by misiRNAs targeting the miRNA seed region was specific for the target mRNA, and misiRNAs targeting 1 nt upstream of miRNA seed region were similarly potent. We also systematically screened the 3'UTR of two mRNA targets using siRNA-tiling experiments. 5' and 3' regions of the p21-activated kinase 6 (PAK6) 3'UTR were found accessible, whereas the middle portion was largely inaccessible for siRNA knockdown. In ribosomal protein S6 kinase (RPS6KB1) 3'UTR, however, only the 5' region was accessible for siRNA knockdown. Detailed analysis of 10 further "difficult-to-silence" targets revealed that siRNA accessibility at the mRNA 3' end is not a general phenomenon, at least in "difficult-to-silence" targets, as we could not detect significant knockdown by siRNAs directed against this region.