Exome Sequencing Starting from Single Cells.

Single-cell genomic analysis enables researchers to gain novel insights across diverse research areas, including developmental biology, tumor heterogeneity, and disease pathogenesis. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging as the amount of genomic DNA present in a single cell is limited. Advancements in multiple displacement amplification (MDA) technologies allow the unbiased amplification of limited quantities of DNA under conditions that maintain its integrity.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.