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Bioline Scholar Monthly: April 2012 Roundup – Breast Cancer

By Bioline: The PCR Company 8 May 2012 No comments

Focus on Breast Cancer

For years scientists have been treating breast cancer as a single disease. However, a new landmark study published in Nature has reclassified breast cancer into ten separate sub-diseases based on their genetic fingerprint. The culmination of decades of research, the study is the largest global study of breast cancer tissue ever performed.

The team, led by the British Columbia Cancer Center in Canada and the Cambridge Cancer Research Institute in the UK, used genome-wide microarrays to analyze the DNA and RNA of 2,000 tumor samples taken from women diagnosed with breast cancer. This huge pool of genetic information (copy number variants, SNPs and gene expression data), as well as survival data, allowed researchers to spot new and previously unacknowledged patterns for ten subtly different cancers that have, historically, been considered as one.

The challenge now is to understand the genetic drivers behind these newly discovered breast cancer variants and to develop new targeted therapies in the future. It could also lead to women with the best prognosis being spared side-effects of chemotherapy. The classification system will likely also form the basis for newer and better ways to diagnose and manage the disease.

Bioline offers a number of reagents that have helped further the study of cancers and, more specifically, breast cancer. So this edition of Bioline Scholar Monthly focuses on the use of Bioline reagents and kits in the field of breast cancer research.

SensiMix SYBR & Fluorescein Kit

In a diverse cohort of breast cancer patients with a 1–5 year tumor relapse versus those with up to 7 years relapse-free survival, RNA was extracted and subjected to microarray and real-time RT-PCR analysis. Among the 299 genes, five genes which included B cell response genes were found to predict with >85% accuracy relapse-free survival. Real-time RT-PCR confirmed the 5-gene prognostic signature that was distinct from an FDA-cleared 70-gene signature of MammaPrint panel and from the Oncotype DX recurrence score assay panel.

Ascierto, L. M., et al. Breast Can. Res. Treat. 131(3):871-880 (2012) - A signature of immune function genes associated with recurrence-free survival in breast cancer patients.

TRIsure reagent

MicroRNAs (miRNAs) are noncoding RNAs that function as key posttranscriptional regulators of gene expression. This paper found that BRCA1 recognizes the RNA secondary structure and directly binds with primary transcripts of miRNAs via a DNA-binding domain. The findings indicate novel functions of BRCA1 in miRNA biogenesis, which may be linked to its tumor suppressor mechanism and maintenance of genomic stability.

Kawai S. and Amano A. J. Cell Biol. 197 (2):201-208 (2012) - BRCA1 regulates microRNA biogenesis via the DROSHA microprocessor complex.

Bioscript

The bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P4) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. The magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P4 may have an important role in breast cancer progression.

Long J. S., et al. J. Biol. Chem. 285:35957-35966 (2010) - Sphingosine 1-Phosphate Receptor 4 Uses HER2 (ERBB2) to Regulate Extracellular Signal Regulated Kinase-1/2 in MDA-MB-453 Breast Cancer Cells.

SensiMix SYBR No-ROX Kit

CD44, the transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. The expression of CD44 and its variants is associated with poor prognosis in breast cancer. This paper investigated the effect of silibinin (a polyphenolic flavonolignan of the herbal plant of Silybum marianum, milk thistle) on the epidermal growth factor (EGF) ligand-induced CD44 expression in human breast cancer cells. The results suggest that silibinin prevents the EGFR signaling pathway and may be used as an effective drug for the inhibition of metastasis of human breast cancer.

Kim S., et al. Anticancer Res. 31(11): 3767-3773 (2011) - Silibinin Suppresses EGFR Ligand-induced CD44 Expression through Inhibition of EGFR Activity in Breast Cancer Cells.

Human papillomavirus (HPV) and Epstein Barr virus (EBV) have been found in breast carcinomas around the world. In this study, fifty-five BCs from Chile were analyzed for HPV and EBV presence. In addition, HPV- 16 viral load/physical status and E6/E7 expressions were determined. The results suggest that it is unlikely that HPV and/or EBV play a direct role in the etiology of breast carcinomas.

Aguayo F., et al. Infectious Agents and Cancer 6:7 (2011) - Human papillomavirus and Epstein-Barr virus infections in breast cancer from chile.

cDNA Synthesis Kit

This study suggests that melatonin may play a role in the desmoplastic reaction in breast cancer through a down regulatory action on the expression of antiadipogenic cytokines, which decrease the levels of these cytokines. Lower levels of cytokines stimulate the differentiation of fibroblasts and decrease both aromatase activity and expression, thereby reducing the number of estrogen-producing cells proximal to malignant cells.

Alonso-González C., et al. J. Pineal Res. 52(3): 282–290, (2012) - Melatonin interferes in the desmoplastic reaction in breast cancer by regulating cytokine production.

Melatonin reduces the development of breast cancer interfering with oestrogen-signalling pathways, and also inhibits aromatase activity and expression. This study shows that melatonin inhibits aromatase activity and expression by regulating the gene expression of specific aromatase promoter regions. A possible mechanism for these effects would be the regulation by melatonin of intracellular cAMP levels, mediated by an inhibition of cyclooxygenase activity and expression.

Martínez-Campa C., et al. British J. Can. 101: 1613–1619 (2009) - Melatonin inhibits aromatase promoter expression by regulating cyclooxygenases expression and activity in breast cancer cells.

IMMOLASE DNA Polymerase

Bisphenol A (BPA) has long been suspected to promote carcinogenesis, but the high doses of BPA used in many studies generated conflicting results. This paper shows that BPA at environmentally relevant doses reduces the efficacy of chemotherapeutic agents. These data provide considerable support to the accumulating evidence that BPA is hazardous to human health.

LaPensee E. W., et al. Environ Health Perspect. 117(2): 175–180 (2009) - Bisphenol A at Low Nanomolar Doses Confers Chemoresistance in Estrogen Receptor-a–Positive and –Negative Breast Cancer Cells.

BIOTAQ DNA Polymerase

This paper indicates that decreased CDH1, CDH13 and TIMP3 with increased CD44 gene expression levels can be used as an indicator for invasion in both ER-positive and ER-negative breast tumors. In double-negative tumor tissues, CD44 can be considered a marker for aggressive properties. However, additional assays in a larger series of patients with long follow up will be necessary to confirm these results of gene expressions in ER-positive and ER-negative tumors and their relationship with HER2 and ESR1.

Celebiler A., et al. Can. Sci. 100: 2341–2345. (2009) - Predicting invasive phenotype with CDH1, CDH13, CD44, and TIMP3 gene expression in primary breast cancer.

Bioline Scholar Monthly: February 2012 Round-Up

By Bioline: The PCR Company 9 March 2012 No comments

It's time for the second in our series of Bioline Scholar Monthly compilations. This month we're focusing on the many uses of Bioline polymerases in forensic science.

Identification of a report's species is one of the basic analyses in forensics. However, due to the nature of the sampling environment, DNA samples often contain PCR-inhibitory substances which may generate blank or incomplete DNA profiles. The common approach to overcoming PCR inhibition is extensive DNA purification, but this can increase the risk of DNA loss. In some cases, isolation of single cells using laser-capture microdissection can be used, but again this reduces the amount of DNA available.

DNA polymerases that can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition, without any additional sample preparation, are therefore advantageous, as are polymerases that result in high yields.

Bioline’s DNA polymerases are very robust and have been carefully designed to overcome these problems. BIO-X-ACT™ Short in particular is specifically designed for difficult/problematic PCR applications that require high processivity and fidelity, applications that would normally fail with other DNA polymerase. MangoTaq™ has also been designed for problematic and ancient DNA, whereas IMMOLASE™ and BIOTAQ™ are high yield for small sample sizes. Together with our new MyTaq™, MyFi™ and RANGER Bioline has polymerases to meet all the high-fidelity requirements of forensic science.

So, without further ado, here's...

Bioline Scholar Monthly: February 2012

BIO-X-ACT™ Short DNA Polymerase

Loop-mediated isothermal amplification (LAMP) is an uncomplicated, quick and relatively inexpensive diagnostic tool. In Barkway and colleagues procedure, BIO-X-ACT Short DNA Polymerase was initially used to successfully verify the LAMP primer pair for Eimeria species specificity using PCR.

Barkway, C. P., et al. BMC Veterinary Research 2011, 7:67 (2011) – Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens

BIO-X-ACT™ Short Mix

In a comparison of nine DNA polymerases, the DNA detection limit was lowest with BIO-X-ACT Short, providing the highest number of improved DNA profiles, using real crime scene saliva samples.

Hedman, J., et al. BioTechniques 47, 951-958 (2009) – Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles

IMMOLASE™ DNA Polymerase

The identification via DNA analysis is reliably and reproducibly possible from well preserved and semi-burnt bones.

Schwark, T., et al Forensic Sci. Int.: Gene. 5(5), 393-399 (2011) – Reliable genetic identification of burnt human remains.

Dried herbarium specimens may be invaluable to understand long-term changes at sites with a history of cyanobacterial blooms.

Metcalf, J.S., et al Harmful Algae 15 47–52 - (2012) – Analysis of microcystins and microcystin genes in 60–170-year-old dried herbarium specimens of cyanobacteria

MangoTaq™ DNA Polymerase

Out of 19 polymerases, the best performance was exhibited by the Mango-Taq DNA polymerase, which was the only polymerase which was able to amplify the ~620 bp amplification product from the 102 year old sample.

Telle, S. & Marco Thines, M. PLoS ONE 3(10):doi:10.1371/journal.pone.0003584 - (2008) – Amplification of cox2 (~620 bp) from 2 mg of Up to 129 Years Old Herbarium Specimens, Comparing 19 Extraction Methods and 15 Polymerases

BIOTAQ™ DNA Polymerase

In Australia and globally, Sarcophagidae flies remain unexploited as indicators of post-mortem interval in forensic investigations. A molecular identification method involving DNA ‘barcoding’ of the mitochondrial COI gene from 16 species of Australian Sarcophagidae was successfully developed. The authors conclude analysis of sarcophagids in forensic entomology should increase and their value as tools in criminal investigations realised.

Meiklejohn, K. A., et al. Int. J. Legal Med. 125(1), 27-32 - (2011) – DNA-based identification of forensically important Australian Sarcophagidae (Diptera)

BIOLASE™ DNA Polymerase

Microsatellite markers were developed for the medicinal plant Tripterygium (Celastraceae) to assess its population structure and to facilitate source tracking of plant materials used for medicinal extracts.

Novy, A. & Jones, K. C. Am. J. Bot. 98(10) e280-e281 (2011) – Characterization of polymorphic microsatellites for Tripterygium (Celastraceae), a monospecific genus of medicinal importance

BIOMIX™

Lindgren funnel traps baited with aggregation pheromones are effective tools for monitoring flight activity in the red flour beetle (Tribolium castaneum) and lesser grain borer (Rhyzopertha dominica). But the samples are affected by the preservation method.

Stevens, M. M,. et al. J. Stored Products Res. 47(2), 69–75 - (2011) – Maintaining DNA quality in stored-grain beetles caught in Lindgren funnel traps

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