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Multiplex quantitative polymerase chain reaction (PCR) based on novel design of fluorescent primers is described. Self-quenched fluorogenic primers are labeled with a single fluorophore on a base close to the 3'-end with no quencher required. A tail of 5-7 nucleotides is added to the 5'-end of the primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial fluorescence of the primers that increases upon formation of the PCR product. The hairpin oligonucleotides (DeltaG from -1.6 to -5.8 kcal/mol) are as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Self-quenched primers could be designed manually or by specialized software and could be used for real-time gene quantitation. Targets of 10-107 copies could be detected with precision in PCR using fluorescein-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method could also be used to detect single nucleotide polymorphism by allele-specific PCR. In conclusion, self-quenched primers are an efficient and cost-effective alternative to fluorescence resonance energy transfer-labeled oligonucleotides.

The TIG3 gene is a retinoic acid inducible class II tumor suppressor gene downregulated in several human tumors and malignant cell lines. Diminished TIG3 expression correlates with decreased differentiation whereas forced expression of TIG3 suppresses oncogenic signaling pathways and subsequently induces differentiation or apoptosis in tumor cells. Analysis of TIG3 mRNA expression in a large set of cDNA pools derived from matched tumor and normal human tissues showed a significant downregulation of TIG3 in 29% of the cDNA samples obtained from ovarian carcinomas. Using in situ hybridization, we demonstrated expression of TIG3 in the epithelial lining of 7 normal ovaries but loss of TIG3 expression in 15/19 of human ovarian carcinoma tissues. In SKOV-3, CAOV-3 and ES-2 ovarian carcinoma cell lines, downregulation of TIG3 mRNA was reversible and dependent on an activated MEK-ERK signaling pathway. Re-expression of TIG3 mRNA in these cells upon specific interference with the MEK-pathway was correlated with growth inhibition of the cells. In OVCAR-3 and A27/80 ovarian carcinoma cells, TIG3 suppression is MEK-ERK independent, but expression could be reconstituted upon interferon gamma (IFNγ) induction. Overexpression of TIG3 in A27/80 ovarian carcinoma cells significantly impaired cell growth and despite increased mRNA levels, TIG3 protein was hardly detectable. These results suggest that TIG3 is negatively regulated by an activated MEK-ERK signaling pathway. Further mechanisms must interfere with TIG3 expression that are independent of MEK and partially include interferon-responsive components. © 2005 Wiley-Liss, Inc.

We studied fluorescence intensity, polarization and lifetime of some commonly used fluorophores conjugated to oligodeoxyribonucleotides with different primary and secondary structures. We found that fluorescence intensity can increase or decrease upon hybridization of the labeled strand to its complement depending on the sequence and position of the fluorophore. Up to 10-fold quenching of the fluorescence upon hybridization was observed when the dye moiety was attached close to the 3' end and the 3'-terminal base was either dG or dC. No quenching upon hybridization was observed when the dye was positioned within the same sequence context but close to the 5' end. The presence of a dG overhang quenches the fluorescence less efficiently than a blunt end dG-dC or dC-dG base pair. When located internally in the double strand, the dG-dC base pair does not affect the fluorescence of the nearby dye. Guanosine in a single-stranded oligonucleotide quenches the fluorescence of nearby dye by

Multiplex quantitative PCR based on novel design of fluorescent primers is described. Fluorogenic primers are labeled with a single fluorophore on a base close to the 3' end with no quencher required. A tail of 5-7 nt is added to the 5' end of the primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial fluorescence of the primers that increases up to 8-fold upon formation of the PCR product. The hairpin oligonucleotides (DeltaG from -1.6 to -5.8 kcal/mol) may be as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Multiple fluorogenic primers were designed by specialized software and used for real-time quantitation of c-myc and IL-4 cDNAs in the presence of reference genes such as beta-actin, GAPDH and 18S rRNA. Targets of 10-10(7) copies were detected with precision in PCR using FAM-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method was also used to detect single nucleotide polymorphism of the human retinal degeneration gene by allele-specific PCR with end-point detection using a fluorescent plate reader or a UV-transilluminator. We conclude that fluorogenic mono-labeled primers are an efficient and cost-effective alternative to FRET-labeled oligonucleotides.

We demonstrate an organic semiconductor distributed feedback (DFB) laser as excitation source in surface-enhanced Raman spectroscopy (SERS). SERS-active substrates comprising nanopillar arrays were fabricated by the technique of laser-assisted nano-replication.

Rho GTPases orchestrate signaling pathways leading to cell migration. Their function depends on GTP loading and isoprenylation by geranylgeranyl pyrophosphate (GGpp). In this study, we show that in human T cells, geranylgeranylation—and not GTP loading—is necessary for RhoA-mediated downstream events. As a result of GGpp depletion with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin, RhoA was sequestered from the membrane to the cytosol and, notwithstanding increased GTP loading, the constitutive activation of its substrate Rho-associated coiled-coil protein kinase-1 was blocked. In line with this, T cells expressing increased GTP-RhoA failed to form an intact cytoskeleton and to migrate toward a chemokine gradient. In vivo treatment with atorvastatin in the rodent model of multiple sclerosis markedly decreased the capacity of activated T cells to traffic within the brain, as demonstrated by multiphoton analysis. Thus, tethering of RhoA to the membrane by GGpp is determinant for T cell migration and provides a mechanism for preventing T cell infiltration into inflamed compartments by 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors

Extracellular vesicles (EVs) play a pivotal role in cancer progression. However, the majority of functional studies performed so far relies on data acquired in traditional 2D cultures. Because the spatial architecture of tissue is decisive for the cell fate, new cell models to study EV functions in the 3D environment must approximate in vitro models to the physiological conditions. Several models were developed during the last years, which may be suitable to serve as 3D models to study EVs; among them are hydrogels, solid scaffolds, bioreactors, and 3D CoSeedis™ inserts. We present in this chapter a protocol for a 3D cell model based on the 3D CoSeedis™ agarose inserts, allowing for a long-term culture of cells of different origins under serum-free conditions and easy EV recovery. Additionally, information on individual culture conditions in 3D CoSeedis™ for different cell lines, protocols for model evaluation, and quality controls are included. We hope that our suggestions and experience will be useful to carry out EV study under more physiological conditions and contribute to the EV research field's progress.

Anterior gradient 2 (AGR2) is a gene predominantly expressed in mucus-secreting tissues or in endocrine cells. Its expression is drastically increased in tumors including prostate cancer. Here we investigated whether AGR2 transcript levels can be used as a biomarker to detect prostate cancer (PCa). Using a PCR-based approach, we could show that in addition to the wild-type (AGRwt long and short) transcripts, five other AGR2 splice variants (SV) (referred to as AGR2 SV-C, -E, -F, -G and -H) were present in cancer cell lines. In tissue biopsies, SV-H and AGR2wt (short) distinguished between benign and PCa (p ≤ 0.05 n = 32). In urine exosomes, AGR2 SV-G and SV-H outperformed serum PSA. Receiver operating characteristic (ROC) curves showed the highest discriminatory power of SV-G and SV-H in predicting PCa. AGR2 SV-G and SV-H are potential diagnostic biomarkers for the non-invasive detection of PCa using urine exosomes.