Amplification efficiency and thermal stability of qPCR instrumentation: Current landscape and future perspectives

This is a final draft version of the publication following acceptance by the journal.Quantitative polymerase chain reaction (qPCR) is a method of amplifying and detecting small samples of genetic material in real time and is in routine use across many laboratories. Speed and thermal uniformity, two important factors in a qPCR test, are in direct conflict with one another in conventional peltier‑driven thermal cyclers. To overcome this, companies are developing novel thermal systems for qPCR testing. More recently, qPCR technology has developed to enable its use in point‑of‑care testing (POCT), where the test is administered and results are obtained in a single visit to a health provider, particularly in developing countries. For a system to be suitable for POCT it must be rapid and reliable. In the present study, the speed and thermal uniformity of four qPCR thermal cyclers currently available were compared, two of which use the conventional peltier/block heating method and two of which use novel heating and cooling methods

Differential effects of rapalogues, dual kinase inhibitors on human ovarian carcinoma cells in vitro

Ovarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7,000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer has a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. Using a wound healing assay we show that inhibition of the mTOR pathway using rapamycin, rapalogues, resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 h in these cell lines. We extended these findings up to 72 h with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer

Differential expression of mTOR components in endometriosis and ovarian cancer: Effects of rapalogues and dual kinase inhibitors on mTORC1 and mTORC2 stoichiometry

© Rogers‑Broadway et al. Endometriosis is a well-known risk factor for ovarian cancer. The genetic changes that characterise endometriosis are poorly understood; however, the mechanistic target of rapamycin (mTOR) pathway is involved. In this study, we investigated the expression of key mTOR components in endometriosis and the effects of rapalogues using an endometrioid ovarian carcinoma cell line (MdAH 2774) as an in vitro model. Gene expression of mTOR, dEPTOR, Rictor and Raptor was assessed by qPcR in 24 endometriosis patients and in silico in ovarian cancer patients. Furthermore, the effects of Rapamycin, Everolimus, deforolimus, Temsirolimus, Resveratrol, and BEZ235 (dactolisib, a dual kinase inhibitor) on mTOR signalling components was assessed. mTOR showed a significant increase in the expression in endometriosis and ovarian endometrioid adenocarcinoma patients compared to non-affected controls. dEPTOR, an inhibitor of mTOR, was downregulated in the advanced stages of ovarian cancer (III and IV) compared to earlier stages (I and II). Treatment of MdAH-2774 cells with the mTOR inhibitors resulted in the significant upregulation of dEPTOR mRNA, whereas treatment with rapamycin and BEZ-235 (100 nM) resulted in downregulation of the mTOR protein expression after 48 h of treatment. None of the treat ments resulted in translocation of mTOR from cytoplasm to nucleus. Upregulation of dEPTOR is a positive prognostic marker in ovarian cancer and is increased in response to mTOR pathway inhibition suggesting that it functions as a tumour suppressor gene in endometrioid ovarian carcinoma. collectively, our data suggest the mTOR pathway as a potential connection between endometriosis and ovarian cancer and may be a potential target in the treatment of both conditions