|Low-volume on-chip single sperm cell analysis|
Pickrahn I. E. ,Schmidt-Gann G., Kroneis T.
We performed low-volume on-chip DNA typing of single sperm cells isolated by means of laser microdissection. 16plex PCR was successful in 39 of 40 single cell samples yielding a mean PCR efficiency of 62.8%. In addition, we were able to identify a single-cell sample containing more than one cell enabling us to monitor the quality of the whole procedure, and hence, exclude “contaminated” samples from further analysis.
|Development and NDA-Level Validation of a Real-Time PCR Procedure for Detection and Quantification of Residual E.coli DNA Contamination of Biopharmaceutical Products|
Dan Papa, Pedro J. Morales and Michael D. Sadick
Escherichia coli (E. coli) has been commonly used for the production of biopharmaceuticals. Among the impurities that must be monitored in biopharmaceuticals is residual host-cell DNA (HCD). This study describes the development and subsequent NDA-level validation of a real time PCR procedure developed in response to a client’s need to improve the sensitivity of detection and quantification of residual E. coli HCD in their drug product.
|Chemically Modified Primers for PCR and Ligation Applications|
Elena Hidalgo Ashrafi, Sabrina Shore, Tony Le, Victor Timoshchuk, Natasha Paul, Richard Hogrefe, Inna Koukhareva, Alexandre Lebedev
PCR is an essential tool with utility in a variety of advanced applications. To improve the specificity of PCR, a unique approach to "Hot Start" PCR employing primers containing thermolabile modifications has been developed. These modified primers, named CleanAmp™ Primers, are amenable for use in Hot Start activation schemes as the modification is released after an initial denaturation step.
|Development of a Test Battery for Epigenetic Non-genotoxic Carcinogens|
The main objective of this sudy is to obtain an increased insight into the mechanisms of action of epigenetic carcinogens. Although the expected number of non-genotoxic carcinogens among newly registered compounds is unknown, there is a growing concern that when numbers of 2-year cancer bioassays are significantly reduced, non-genotoxic carcinogens may go undetected. Therefore there is a need for the development of alternative methods for their detection.
|Chemically Modified Primers for Improved Multiplex PCR|
Elena Hidalgo Ashrafi, Tony Le, Alexandre Lebedev, Richard Hogrefe, Victor Timoshchuk, Sabrina Shore, Inna Koukhareva and Natasha Paul
Multiplex PCR is an advantageous technique used in PCR applications to amplify multiple targets in a single reaction. As useful as it is, this technique presents a new set of challenges that further complicates PCR setup. For example, reactions are more prone to off-target amplifications such as mis-priming and primer dimer due to the increased number of primer pairs.
|Hot Start dNTPs - Novel Chemistries for Use in Advanced PCR Applications|
Tony Le, Elena Hidalgo Ashrafi, Sabrina Shore, Victor Timoshchuk, Natasha Paul, Richard Hogrefe, Inna Koukhareva, Alexandre Lebedev
PCR is a widely used scientific tool whose specificity can be increased by the use of Hot Start technologies. Although many Hot Start technologies exist, recently developed CleanAmp™ dNTPs are a distinct approach that employs modified nucleoside triphosphates with a thermolabile protecting group at the 3´-hydroxyl.
|ROLE OF IRF-8 IN THE INTERFACE OF MELANOMA TUMOR CELL-IMMUNE SYSTEM INTERACTION.|
Fabrizio Mattei, Giovanna Schiavoni, Massimo Spada, Francesca Spadaro and Lucia Gabriele
Interferon-regulatory factor-8 is essential for differentiation and function of dendritic cells and thus for the induction of competent immune responses. Here, we investigated the role of IRF-8 in affecting immune response against melanoma. Our results reveal a critical role of IRF-8 in controlling melanoma growth and suggest that IRF-8-mediated antitumor activity is the result of a coordinated action between DC-mediated immune response and the tumor suppressor function of IRF-8.
|NEW TECHNOLOGIES FOR FFPE SAMPLES: Improved RNA Isolation and novel cDNA priming for qPCR and for universal mRNA amplification|
G Krupp1; R Jaggi2; D Englert3, DJ Wilson3, S Laken3, S, ES Quabius4
Improved FFPE RNA isolation, novel TR priming for complete recovery of FFPE mRNA fragments. Differential expression defines Scores for tumor classification: comparing fresh-frozen with FFPE. Novel flow-through Ziplex microarray platform demonstrates MAQC performance level of data obtained with FFPE samples.
|FOXP3 Gene Expression in Multiple Sclerosis patients before and after Mesenchymal Stem Cell therapy|
Maryam Mohajeri, Mandana Mohyeddin Bonab, Behrooz Nikbin, Ali Farazmand
Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disorder of the CNS. No successful treatment for MS, but one therapeutic strategy in research is the use of bone marrow-derived mesenchymal stem cells (MSC). We studied a group of MS patients who underwented MSCs, assayed for expression of a transcription factor, FOXP3, as a specific marker of MS amelioration in peripheral blood. qRT-PCR on PBMCs showed higher FoxP3 levels.