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1.  No evidence for promoter region methylation of the succinate dehydrogenase and fumarate hydratase tumour suppressor genes in breast cancer 
BMC Research Notes  2009;2:194.
Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes that are also known to act as tumour suppressor genes. Increased succinate or fumarate levels as a consequence of SDH and FH deficiency inhibit hypoxia inducible factor-1α (HIF-1α) prolyl hydroxylases leading to sustained HIF-1α expression in tumours. Since HIF-1α is frequently expressed in breast carcinomas, DNA methylation at the promoter regions of the SDHA, SDHB, SDHC and SDHD and FH genes was evaluated as a possible mechanism in silencing of SDH and FH expression in breast carcinomas.
No DNA methylation was identified in the promoter regions of the SDHA, SDHB, SDHC, SDHD and FH genes in 72 breast carcinomas and 10 breast cancer cell lines using methylation-sensitive high resolution melting which detects both homogeneous and heterogeneous methylation.
These results show that inactivation via DNA methylation of the promoter CpG islands of SDH and FH is unlikely to play a major role in sporadic breast carcinomas.
PMCID: PMC2760567  PMID: 19778456
2.  Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays 
BMC Research Notes  2009;2:112.
The development of reverse transcription – quantitative real-time PCR (RT-qPCR) platforms that can simultaneously measure the expression of multiple genes is dependent on robust assays that function under identical thermal cycling conditions. The use of a primer optimisation matrix to improve the performance of RT-qPCR assays is often recommended in technical bulletins and manuals. Despite this recommendation, a comprehensive introduction to and evaluation of this approach has been absent from the literature. Therefore, we investigated the impact of varying the primer concentration, leaving all the other reaction conditions unchanged, on a large number of RT-qPCR assays which in this case were designed to be monitored using hydrolysis probes from the Universal Probe Library (UPL) library.
Optimal RT-qPCR conditions were determined for 60 newly designed assays. The calculated Cq (Quantification Cycle) difference, non-specific amplification, and primer dimer formation for a given assay was often dependent on primer concentration. The chosen conditions were further optimised by testing two different probe concentrations. Varying the primer concentrations had a greater effect on the performance of a RT-qPCR assay than varying the probe concentrations.
Primer optimisation is important for improving the performance of RT-qPCR assays monitored by UPL probes. This approach would also be beneficial to the performance of other RT-qPCR assays such as those using other types of probes or fluorescent intercalating dyes.
PMCID: PMC2706253  PMID: 19549292
3.  A new approach to primer design for the control of PCR bias in methylation studies 
BMC Research Notes  2008;1:54.
Primer design for PCR-based methylation analysis following bisulfite conversion of DNA is considerably more complex than primer design for regular PCR. The choice of the optimal primer set is critical to the performance and correct interpretation of the results. Most methodologies in methylation analysis utilize primers that theoretically amplify methylated and unmethylated templates at the same time. The proportional amplification of all templates is critical but difficult to achieve due to PCR bias favouring the amplification of the unmethylated template. The focus of this brief communication is to point out the important criteria needed for the successful choice of primers that will enable the control of PCR bias in bisulfite based methylation-screening protocols.
PMCID: PMC2525644  PMID: 18710507
4.  Detection of the transforming AKT1 mutation E17K in non-small cell lung cancer by high resolution melting 
BMC Research Notes  2008;1:14.
A recurrent somatic mutation, E17K, in the pleckstrin homology domain of the AKT1 gene, has been recently described in breast, colorectal, and ovarian cancers. AKT1 is a pivotal mediator of signalling pathways involved in cell survival, proliferation and growth. The E17K mutation stimulates downstream signalling and exhibits transforming activity in vitro and in vivo.
We developed a sensitive high resolution melting (HRM) assay to detect the E17K mutation from formalin-fixed paraffin-embedded tumours. We screened 219 non-small cell lung cancer biopsies for the mutation using HRM analysis. Four samples were identified as HRM positive. Subsequent sequencing of those samples confirmed the E17K mutation in one of the cases. A rare single nucleotide polymorphism was detected in each of the remaining three samples. The E17K was found in one of the 14 squamous cell carcinomas. No mutations were found in 141 adenocarcinomas and 39 large cell carcinomas.
The AKT1 E17K mutation is very rare in lung cancer and might be associated with tumorigenesis in squamous cell carcinoma. HRM represents a rapid cost-effective and robust screening of low frequency mutations such as AKT1 mutations in clinical samples.
PMCID: PMC2442881  PMID: 18611285

Results 1-4 (4)