Read by QxMD icon Read


J A Sargent, Vhj Roberts, J E Gaffney, A E Frias
Geometry of the placental villous vasculature is a key determinant of maternal-fetal nutrient exchange for optimal fetal growth. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy; however, the methodology lacks a signal:noise ratio of sufficient magnitude to allow for quantitative analysis. Thus, we sought to develop a reproducible method to investigate the 3D vasculature of the nonhuman primate placenta for subsequent data analysis. Fresh placental tissue was dissected, formalin fixed, clarified using a modified Visikol® protocol and immunolabeled for CD31 (fetal endothelium) and cytokeratin-7 (villous trophoblast) for confocal imaging of the microanatomy...
October 29, 2018: BioTechniques
Ibrahim Kays, Brian Edwin Chen
Single-cell analysis overcomes the problems of cellular heterogeneity by revealing the individual differences between cells in tissue. The current tools used to profile gene expression at the single-cell level are arduous and often require specialized equipment. We have previously developed a technique to quantify protein expression levels in single living cells. Here, we combine quantification of protein expression with absolute measurement of mRNA amounts of the same gene in the same cell, to profile the expression of genes at the transcriptional and translational levels...
October 18, 2018: BioTechniques
Arvind Venkataraman, Mirjana Parlov, Ping Hu, Dan Schnell, Xingtao Wei, Jay P Tiesman
Shotgun metagenomics is a powerful platform to characterize human microbiomes. However, to translate such survey data into consumer-relevant products or services, it is critical to have a robust metagenomics workflow. We present a tool - spike-in DNA - to assess performance of metagenomics workflows. The spike-in is DNA from two organisms - Alivibrio fischeri and Rhodopseudomonas palustris, in a ratio of 4:1 added to samples before DNA extraction. With a valid workflow, the output ratio of relative abundances of these organisms should be close to 4...
September 17, 2018: BioTechniques
Pei-Zhong Tang, Bo Ding, Lansha Peng, Vadim Mozhayskiy, Jason Potter, Jonathan D Chesnut
GUIDE-seq was developed to detect CRISPR/Cas9 off-target. However, as originally reported, it was associated with a high level of nonspecific amplification. In an attempt to improve it, we developed target-enriched GUIDE-seq (TEG-seq). The sensitivity level reached 0.1-10 reads-per-million  depending on the NGS platform used, which was equivalent to 0.0002-1% measured by Targeted Amplicon-seq. Application of TEG-seq was demonstrated for the evaluation of various Cas9/gRNA configurations, which suggests delivery of Cas9/gRNA ribonucleoprotein results in significantly fewer off-targets than Cas9/gRNA plasmid...
August 17, 2018: BioTechniques
Sean R Koebley, Jason Reed
No abstract text is available yet for this article.
November 2018: BioTechniques
Alistair R McTaggart, Tuan A Duong, Vang Quy Le, Louise S Shuey, Werner Smidt, Sanushka Naidoo, Michael J Wingfield, Brenda D Wingfield
It is challenging to sequence and assemble genomes of obligate plant pathogens and microorganisms because of limited amounts of DNA, comparatively large genomes and high numbers of repeat regions. We sequenced the 1.2 gigabase genome of an obligate rust fungus, Austropuccinia psidii, the cause of rust on Myrtaceae, with a Chromium 10X library. This technology has mostly been applied for single-cell sequencing in immunological studies of mammals. We compared scaffolds of a genome assembled from the Chromium library with one assembled from combined paired-end and mate-pair libraries, sequenced with Illumina HiSeq...
November 2018: BioTechniques
Bradford W Daigneault, Marcela Vilarino, Sandeep K Rajput, Tristan Frum, George W Smith, Pablo J Ross
CRISPR technologies used for mammalian embryology have wide implications from basic research to applications in agriculture and biomedicine. Confirmation of successful gene editing following CRISPR/Cas9 delivery is often limited to either protein expression or sequencing analyses of embryos but not both, due to technical challenges. Herein we report an integrative approach for evaluating both protein expression and genotype of single embryos from fixed bovine embryos previously subjected to CRISPR/Cas9 microinjection...
November 2018: BioTechniques
Scott G Canfield, Gyuhyung Jin, Sean P Palecek, Tori Sampsell
Cell culture is a vital component of laboratories throughout the scientific community, yet the absence of standardized protocols and documentation practice challenges laboratory efficiency and scientific reproducibility. We examined the effectiveness of a cloud-based software application, CultureTrax® as a tool for standardizing and transferring a complex cell culture protocol. The software workflow and template were used to electronically format a cardiomyocyte differentiation protocol and share a digitally executable copy with a different lab user...
November 2018: BioTechniques
Abigail Sawyer, Joseph Martin
Abigail Sawyer and Joseph Martin investigate how microscopy is evolving in order for cells and processes to be better visualized in their native environments.
November 2018: BioTechniques
Megan M Thompson, Estelle M Hrabak
Whatman FTA® Cards are a fast and efficient method for capturing and storing nucleic acids but can be cost-prohibitive for large numbers of samples. We developed a method that substitutes a readily-available cellulose matrix and homemade washing buffer for commercial FTA® Cards and FTA® Purification Reagent. This method is suitable for long-term storage of DNA from many plant species prior to PCR.
November 2018: BioTechniques
Mark F Kavlick
Described here is a novel, universal exogenous internal positive control (IPC), which is fully synthetic for unparalleled quality control. The IPC was rationally designed, is small and efficiently amplified, has been successfully utilized alone or in triplex qPCR reactions, and is not crossreactive to human DNA or to any of the numerous non-human DNA samples tested. It was sensitive to inhibition by at least four commonly encountered amplification inhibitors. The IPC was used in a hydrolysis probe qPCR assay; however, it may be adaptable for other applications such as intercalating dye qPCR, PCR, isothermal amplification and reverse transcription-PCR...
November 2018: BioTechniques
(no author information available yet)
No abstract text is available yet for this article.
November 2018: BioTechniques
Jing Yi Lai, Qiuting Loh, Yee Siew Choong, Theam Soon Lim
Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains - a chain shuffling process for antibody maturation...
November 2018: BioTechniques
Sarah M Rue, Paul W Anderson, Jessica M Miller, Salvatore G Fanale, Jim Y Chang, Scott M Glaser, Scott A Lesley
High-throughput protein expression platforms are increasingly used to produce proteins for many applications: to support studies in structure/function, regulation and proteomics, as well as for direct use as potential biotherapeutic agents for medical applications. Here we describe a device that we refer to as the flask density reader (FDR) consisting of a through-beam laser and sensor, and a customized culture flask-receiving nest. The FDR has been integrated onto GNF System™'s automated protein expression platform to enable rapid, noninvasive, fully automated spectrophotometric determination of cell densities in suspension mammalian cell cultures...
October 2018: BioTechniques
Jong Won Lee, Jae Sook Sung, Young Soo Park, Seok Chung, Yeul Hong Kim
Isolation of spheroid-forming cells is important to investigate cancer stem cell (CSC) characteristics. However, conventional tumor spheroid culture methods have not proven suitable because the aggregated spheroids generally maintain their original heterogeneity and harbor multiple cells with various characteristics. Here we cultured spheroids using a polydimethylsiloxane microwell-based method and a limiting dilution protocol. We then isolated and enriched for CSCs that formed single cell-derived spheroids from gastric cancer cell lines...
October 2018: BioTechniques
Carmen Santana-Calvo, Francisco Romero, Ignacio López-González, Takuya Nishigaki
Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However, most of the popular FRET pairs suffer cross-excitation of the acceptor, which could lead to false positives. To overcome this problem, we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example, we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide-binding domains and cyclic nucleotides...
October 2018: BioTechniques
Joo Chuan Yeo, Chwee Teck Lim
No abstract text is available yet for this article.
October 2018: BioTechniques
Anita Mäki, Marja Tiirola
Ribosomal RNA analysis is a useful tool for characterization of microbial communities. However, the lack of broad-range primers has hampered the simultaneous analysis of eukaryotic and prokaryotic members by amplicon sequencing. We present a complete workflow for directional, primer-independent sequencing of size-selected small subunit ribosomal RNA fragments. The library preparation protocol includes gel extraction of the target RNA, ligation of an RNA oligo to the 5'-end of the target, and cDNA synthesis with a tailed random-hexamer primer and further barcoding...
October 2018: BioTechniques
Hanyoup Kim, Aaron E Ruby, Harini G Shandilya, Arvind K Virmani, Nandita Rahman, Christina M Strange, Jarkko Huuskonen
We have developed a simple and robust probe-free quantitative PCR (qPCR) assay method that can detect minor mutant alleles with a frequency as low as 0.1% in a heterogeneous sample by introducing a novel T-blocker concept to the allele-specific PCR method. Four new KRAS and BRAF mutation detection assays were developed and their performance was demonstrated by testing a large number of replicates, utilizing a customized PCR protocol. Highly efficient and specific mutant amplification in conjunction with selective wild-type suppression by the T-blocker concept enabled 0...
October 2018: BioTechniques
Francesca Lake
No abstract text is available yet for this article.
October 2018: BioTechniques
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"