|Changes in the Gene Expression of mRNA Transcripts for Insulin like Growth Factor, their receptor and Facilitative Glucose Transporter in IVM Oocytes|
S.C. Gupta, Neelam Gupta and Alok Pandey
The cDNA libraries from single oocytes and pre-implantation buffalo embryos from 2 cell stage to blastocyst were established. Relative expression studied with RT-PCR of IGF-I showed an increase at 12h and decline at 24h of IVM oocytes. IGF-IR was expressed at cleavage stages to blastocyts. Glut-I was expressed in IVM oocytes and SCNT embryos at all stages. Gene expression of IGF-I, IGF-IR and Glut-I plays an important role in SCNT embryo production.
|DNA Methylation Analysis – Reliable Cell Characterization in Regenerative Medicine|
Uli Hoffmueller, Stephen Rapko, Udo Baron, Georg Wieczorek, Alexander Hellwag, Cornelia Krüger, Stefan Kärst, Leslie Wolfe and Sven Olek
We demonstrate that DNA methylation patterns can serve as characteristic markers to distinguish different cell types. We have identified panels of methylation markers that are specific to mesenchymal stem cells or various differentiated cell types in the mesenchymal lineage. This method of cell type identification has a number of advantages over conventional markers in that it is robust, is both qualitative and quantitative.
|EM Algorithm for Gene Copy Number Estimation Using TaqMan® Assays|
Catalin Barbacioru, Kelly Li, Caifu Chen and Raymond Samaha
Recently, TaqMan® assays have been developed for detection of genetic variation at gene level using primers and probes designed for genomic DNA sequences. The R package TaqGCN contains classes and methods that can be used for data reading and plotting, and for predicting gene copy number.
|EasyBeacons™ - new Probes Ideal for Realtime PCR Detection of Methylation Status of Single CpG Duplets and SNPs|
K. Skadhauge, C. Nielsen & U.B. Christensen
The EasyBeacons™ presented here are based on the novel technology Intercalating Nucleic Acid, INA®, linked to a fluorophore and a quencher. INA® is composed of normal DNA nucleotides and Intercalating Pseudo Nucleotides (IPNs). The fact that the EasyBeacons™ are mostly composed of normal DNA nucleotides means that in many respects EasyBeacons™ behave like DNA based probes, allowing use of standard buffers, primers and enzymes and hence reduces the optimisation efforts.
|Real-time PCR Gene Expression Profiling|
Mikael Kubista, José Manuel Andrade, Björn Sjögreen and Amin Forootan
Correct interpretation of real-time PCR data requires appropriate experimental design, accurate data pre-processing and analysis of the data using proper statistical and multivariate methods. For this process we have developed the GenEx software.
|Sensitive and Specific Detection of microRNAs|
Martin Kreutz, James Qin, Holger Engel, Po-Jen Shih, Peter Hahn, Martin Schlumpberger, Subrahmanyam Yerramilli and Eric Lader
MicroRNAs are endogenous, 21–22 nt, noncoding RNAs that mediate post transcriptional gene regulation. miRNAs are involved in regulation of gene expression during development, differentiation, cell proliferation, and apoptosis. Misregulation of miRNA expression is associated with several cancers and other diseases. We have developed the miScript System for real-time PCR analysis of hundreds of miRNAs, sno RNAs, piRNAs, other small noncoding RNAs.
|Real-time PCR in Diagnostics of Plant Viruses |
Mehle N., Boben J. and Ravnikar M.
Sensitive and specific method real-time PCR was developed for diagnostics of certain plant viruses. Real-time PCR can be used for determination of different viruses as well as for differentiation between related strains of viruses. The method can also be used for detection of low concentrations of plant viruses in various samples such as environmental waters, growth substrates and seeds.
|Real-Time Multiplex Rt-PCR on Circulating Tumor Cells|
Anieta M. Sieuwerts, Stefan Sleijfer, Jaco Kraan, Joan Bolt-de Vries, Jan-Willem Gratama, John WM Martens and John A. Foekens
Using the CTC kit (CellSearch™), cells that attached to anti-EpCAM Moab were immunomagnetically separated and used for analysis of a selected pilot set of 32 genes by real time RT-PCR. This study shows the feasibility of multiple gene expression analysis on RNA isolated from only one tumor cell. Most importantly, expression analysis of several tumor-specific genes in blood samples containing only 2 tumor cells is already possible.
|Novel Fluidics Microbead Trap/Flow Cell Enhances Speed/Sensitivity of Bead-Based Bioassays Up to 5-Fold|
RM Ozanich, CJ Bruckner-Lea, JW Grate, MG Warner, BP Dockendorff, KC Antolick, HC Edberg, LH Johnson, AN Easterday
Pacific Northwest National Laboratory (PNNL) has developed a micro/nano particle trap that allows surface-functionalized magnetic or non-magnetic particles to be trapped with subsequent perfusion of sample, reagents and wash solutions, yielding significant (up to 5-fold) improvements in assay speed and sensitivity, while significantly reducing sample matrix effects.