Fenrich J.,Biorad Laboratories
American Laboratory | Year: 2015
Researchers at the Lunenfeld Tanenbaum Research Institute at Mount Sinai Hospital in Toronto assessed the culture conditions that support human embryonic stem cell (hESC) pluripotency. Traditional quantitative reverse transcriptionPCR presented researcher Mark Kibschull with challenges: the need to purify RNA prior to RTqPCR restricted the number of samples that could be processed at one time, and analyzing gene targets for hESC differentiation meant that multiple cell cultures were required to produce sufficient sample for analysis. The first step in the workflow uses a lysis reagent that eliminates the need for RNA purification, reducing potential loss of sample. The RNA lysate generated by the SingleShot Cell Lysis Kit is free of contaminating genomic DNA and used directly in the RTqPCR. By lysing embryoid bodies directly and eliminating the RNA purification step, Kibschull dramatically increased his experimental success rate. Kibschull was still unable to profile all the necessary gene targets to draw meaningful conclusions about the differentiation of the stem cells. The problem was solved by performing target specific cDNA preamplification with BioRad SsoAdvanced PreAmp Supermix, which is formulated to allow 1000-fold enrichment of up to 100 targets in a single reaction. With the PrimePCR hESC panel, Kibschull analyzes markers from many differentiation pathways, rather than only a handful of markers spanning a few representative cell fates. The additional markers allow more thorough characterization of the response of stem cell lines to changes in culture conditions.
Descamps V.K.,Biorad Laboratories
American Laboratory | Year: 2015
Neuroscientists realized that the only way to know whether a rat's brain is injected or not, was to use a fluorescent reporter and image slices of the brain. Jody Martin, Ph.D., lab director of the Cell & Molecular Physiology Department at Loyola University Medical Center from Maywood, Ill., US, had to confirm that his viral injection experiments in rat brains targeted the right domain, and that the virus successfully infected the desired neurons. He also needed to share these results with a collaborator to continue with the next step in the experiment, immunostaining the sections.
Sze M.A.,University of British Columbia |
Abbasi M.,Biorad Laboratories |
Hogg J.C.,University of British Columbia |
Sin D.D.,University of British Columbia
PLoS ONE | Year: 2014
Background: Low biomass in the bacterial lung tissue microbiome utilizes quantitative PCR (qPCR) 16S bacterial assays at their limit of detection. New technology like droplet digital PCR (ddPCR) could allow for higher sensitivity and accuracy of quantification. These attributes are needed if specific bacteria within the bacterial lung tissue microbiome are to be evaluated as potential contributors to diseases such as chronic obstructive pulmonary disease (COPD). We hypothesize that ddPCR is better at quantifying the total bacterial load in lung tissue versus qPCR. Methods: Control (n = 16) and COPD GOLD 2 (n = 16) tissue samples were obtained from patients who underwent lung resection surgery, were cut on a cryotome, and sections were assigned for use in quantitative histology or for DNA extraction. qPCR and ddPCR were performed on these samples using primers spanning the V2 region on the 16S rRNA gene along with negative controls. Total 16S counts were compared between the two methods. Both methods were assessed for correlations with quantitative histology measurements of the tissue. Results: There was no difference in the average total 16S counts (P>0.05) between the two methods. However, the negative controls contained significantly lower counts in the ddPCR (0.55 ± 0.28 16S/uL) than in the qPCR assay (1.00 ± 0.70 16S copies) (P <0.05). The coefficient of variation was significantly lower for the ddPCR assay (0.18 ± 0.14) versus the qPCR assay (0.62 ± 0.29) (P<0.05). Conclusion: Overall the ddPCR 16S assay performed better by reducing the background noise in 16S of the negative controls compared with 16S qPCR assay. © 2014 Sze et al.
Rusniok C.,Institute Pasteur Paris |
Couve E.,Institute Pasteur Paris |
Da Cunha V.,Institute Pasteur Paris |
El Gana R.,Institute Pasteur Paris |
And 7 more authors.
Journal of Bacteriology | Year: 2010
Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is an increasing cause of endocarditis among streptococci and frequently associated with colon cancer. S. gallolyticus is part of the rumen flora but also a cause of disease in ruminants as well as in birds. Here we report the complete nucleotide sequence of strain UCN34, responsible for endocarditis in a patient also suffering from colon cancer. Analysis of the 2,239 proteins encoded by its 2,350-kb-long genome revealed unique features among streptococci, probably related to its adaptation to the rumen environment and its capacity to cause endocarditis. S. gallolyticus has the capacity to use a broad range of carbohydrates of plant origin, in particular to degrade polysaccharides derived from the plant cell wall. Its genome encodes a large repertoire of transporters and catalytic activities, like tannase, phenolic compounds decarboxylase, and bile salt hydrolase, that should contribute to the detoxification of the gut environment. Furthermore, S. gallolyticus synthesizes all 20 amino acids and more vitamins than any other sequenced Streptococcus species. Many of the genes encoding these specific functions were likely acquired by lateral gene transfer from other bacterial species present in the rumen. The surface properties of strain UCN34 may also contribute to its virulence. A polysaccharide capsule might be implicated in resistance to innate immunity defenses, and glucan mucopolysaccharides, three types of pili, and collagen binding proteins may play a role in adhesion to tissues in the course of endocarditis. Copyright © 2010, American Society for Microbiology. All Rights Reserved.