Steven T. Okino's research while affiliated with Bio-Rad Laboratories and other places
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Publications (20)
RNA‐Seq is a powerful technique that measures the presence and abundance of RNAs in a cellular sample. There are two general RNA‐Seq approaches. Standard RNA‐Seq processes long RNAs, typically larger than 200 bases. Small RNA‐Seq assesses RNAs shorter than 200 bases and often targets miRNAs in the 20–26 base range. Neither of these technologies cap...
We have developed a highly sensitive RT‐qPCR‐based workflow that quantifies the expression of lncRNAs in a RNA sample. The workflow utilizes a novel reagent that enables streamlined genomic DNA removal, cDNA synthesis and multi‐target pre‐amplification and allows for reliable quantification of as little as 25 pg of RNA (single cell equivalent). Thi...
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated endonuclease Cas9 have emerged as a revolutionary genome engineering tool. Using CRISPR/Cas9 to induce mutations in protein encoding genomic DNA regions is a powerful method for studying protein function in the native cellular context. However, the existing workfl...
Single cell gene expression analysis is a powerful technique that provides a unique and insightful perspective on biological pathways and processes. Here we present a robust workflow that enables fast and accurate analysis of up to 100 genes in isolated single cells. Our workflow is highly sensitive, by assessing RNA reference standards we find tha...
Single cell gene expression analysis is a powerful technique that provides a unique and insightful perspective on biological pathways and processes. Here we present a robust workflow that enables fast and accurate analysis of up to 100 genes in isolated single cells. Our workflow is highly sensitive, by assessing RNA reference standards we find tha...
We developed a novel PCR-based pre-amplification (PreAmp) technology that can increase the abundance of over 350 target genes one million-fold. To assess potential bias introduced by PreAmp we utilized ERCC RNA reference standards, a model system that quantifies measurement error in RNA analysis. We assessed three types of bias: amplification bias,...
Transcriptional profiling is a powerful tool to study biological mechanisms during stem cell differentiation and reprogramming. Genome-wide methods like microarrays or next generation sequencing are expensive, time consuming, and require special equipment and bioinformatics expertise. Quantitative RT-PCR remains one of today's most widely accepted...
We developed a novel PCR-based pre-amplification (PreAmp) technology that can increase the abundance of over 350 target genes one million-fold. To assess potential bias introduced by PreAmp we utilized ERCC RNA reference standards, a model system that quantifies measurement error in RNA analysis. We assessed three types of bias: amplification bias,...
We developed a novel PCR‐based pre‐amplification (PreAmp) technology that can increase the abundance of over 350 target genes one million‐fold. To assess bias introduced by PreAmp we utilized ERCC RNA reference standards, a model system that quantifies measurement error in RNA analysis workflows. We assessed three types of bias: amplification bias,...
Filtering small nucleic acids using permeabilized cells and methods for using the filtering to detect genomic DNA accessibility are described.
The present invention provides compositions and methods for purifying RNA-free DNA from a sample.
Recently we developed novel technology for isolating accessible chromatin. When cells are permeabilized and treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin can be easily isolated and characterized. These DNA fragments, when analyzed by next-generation sequencing, provide a global map of accessible chromatin...
The activity of DNA regulatory elements are often controlled by chromatin structure. Active regulatory elements are located within “open” or “accessible” chromatin. If a regulatory element is in a “closed” or “inaccessible” chromatin environment it is functionally silenced. We find that when permeabilized cells are treated with nuclease in situ, sm...
When permeabilized cells are treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin, can be readily isolated. These small DNA fragments, when analyzed by next‐gen sequencing, provide a global map of accessible chromatin. Here we report the application of this technology in identifying chromatin domains involved in...
We have developed a novel technology for isolating accessible chromatin. When cells are permeabilized and treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin can be easily isolated and characterized. We now report that the isolated accessible chromatin is still associated with its cognate DNA‐binding proteins a...
Citations
... However, "background noise" has to be expected when using whole blood because the whole blood might comprise tumor-specific biomarkers, among many others. Nevertheless, due to the complex isolation procedure and the low RNA yield from exosomes, pre-amplification is often necessary for subsequent gene expression analysis, despite the risk of amplification-induced bias [21,28,29]. ...
... Cultures could be passaged at 1:4 ratios every 3-4 weeks and maintained for more than 135 days (passage 5). For RNA analysis, one 12-well was lysed in RNeasy Plus buffer (Qiagen, Canada) and processed as described 20 . For immunocytochemistry, one 12-well was split 1:8 into 4-well plates (Nunc, Canada), cultured for 7 days and fixed for double-immunocytochemistry using myogenic lineage markers and appropriate secondary antibodies. ...
