Lanoix D.,University of Québec |
Lanoix D.,Biomedical Research Center |
Lacasse A.,University of Québec |
Lacasse A.,Biomedical Research Center |
And 7 more authors.
Molecular Biotechnology | Year: 2012
ReversEtranscription quantitative polymerase chain reaction (RT-qPCR) is a rapid and high throughput gene expression quantification technology. In order to obtain accurate results, several key experimental design and standardization steps must be rigorously followed as previously described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. This study investigates the effect of reference gene normalization and the impact of RNA degradation on gene expression of 8-oxoguanine DNA glycosylase in human placenta from pregnancies complicated by preeclampsia and gestational diabetes mellitus and their gestation-matched controls. The data presented here show how RNA quality and appropriate reference gene selection is not only important to obtain accurate and reproducible RT-qPCR data but how different and even opposite results can be reported if the key steps outlined in the MIQE guidelines are not followed. The procedures and associated results presented in this study provide the first practical application of the MIQE guidelines to placental analysis in normal and pathological pregnancies. © Springer Science+Business Media, LLC 2012.
Thirukkumaran C.M.,University of Calgary |
Thirukkumaran C.M.,Tom Baker Cancer Center |
Nodwell M.J.,Bio Rad Laboratories Canada Ltd. |
Hirasawa K.,Memorial University of Newfoundland |
And 19 more authors.
Cancer Research | Year: 2010
Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor. ©2010 AACR.
Wan A.M.D.,University of Toronto |
Sadri A.,Bio Rad Laboratories Canada Ltd. |
Young E.W.K.,University of Toronto
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2015
We report a novel method for achieving consistent liquid phase solvent bonding of plastic microfluidic devices via the use of retention grooves at the bonding interface. The grooves are patterned during the regular microfabrication process, and can be placed at the periphery of a device, or surrounding microfluidic features with open ports, where they effectively mitigate solvent evaporation, and thus substantially reduce poor bond coverage. This method is broadly applicable to a variety of plastics and solvents, and produces devices with high bond quality (i.e., coverage, strength, and microfeature fidelity) that are suitable for studies in physics, chemistry, and cell biology at the microscale. © The Royal Society of Chemistry 2015.
Konstantinou D.,University of Toronto |
Shirazi A.,University of Toronto |
Sadri A.,Bio Rad Laboratories Canada Ltd. |
Young E.W.K.,University of Toronto
Sensors and Actuators, B: Chemical | Year: 2016
Current methods for fabricating thermoplastic microfluidic devices are either appropriate for prototyping in research labs, or for mass production for commercialization purposes. However, methods for fabricating plastic microfluidic devices in medium volume production have not been demonstrated, and thus, creates a gap in manufacturing methods. A medium volume manufacturing method would be practical and necessary during the technology development phase when many fabricated devices may be required for testing and validation, but design modifications may still be required, thereby making mass production unnecessary and not economical. This study demonstrates the feasibility of combining hot embossing and milling methods to achieve medium volume production of thermoplastic microfluidic devices with high replication fidelity. Hot embossing parameters were tested and optimized on two different embossing systems and for two different plastics (COP and PMMA). Using these optimal hot embossing parameters together with improvements in epoxy mold fabrication and a streamlined milling procedure, a production rate of ∼50 devices per week was achieved during a proof-of-concept medium volume production run. Importantly, this approach also provides a flexible strategy that can accommodate prototype design changes without resulting in long development delays. Overall, results provide evidence that medium production levels can be performed in research labs and in industry, thereby providing an accelerated path to commercialization. © 2016 Elsevier B.V. All rights reserved.
Taylor S.C.,Bio Rad Laboratories Canada Ltd. |
Berkelman T.,Bio Rad Laboratories Inc. |
Yadav G.,Bio Rad Laboratories Inc. |
Hammond M.,Bio Rad Laboratories Inc.
Molecular Biotechnology | Year: 2013
Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In order to have confidence in densitometric data from western blots, researchers should be able to demonstrate statistically significant fold differences in protein expression. This entails a necessary evolution of the procedures, controls, and the analysis methods. We describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods. © 2013 The Author(s).
Taylor S.,Bio Rad Laboratories Canada Ltd.
American Laboratory | Year: 2013
The publication of the MIQE (Minimum Information for the publication of Quantitative real-time PCR Experiments) guidelines provided the scientific community with criteria to help achieve high-quality RT-qPCR (reverse transcription quantitative polymerase chain reaction). Researchers should take care to ensure that the differences in gene expression reflect the different treatments or underlying biological differences and are not a direct result of flawed execution. The surgical removal of tissue, or the collection of cells from a plate, can be stressful when the researcher has not had adequate training and practice. Bearing in mind that the transcriptome is dynamic and highly sensitive to environmental factors, the methodology to harvest the sample must be highly reproducible in the shortest possible time frame. All self-designed assays must be vetted through an in-depth bioinformatics pipeline to ensure optimal performance.