Bioline Scholar Monthly: April 2012 Roundup – Breast Cancer

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.

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.

Bioline Celebrates 20 Years of Service to Life Science

How time flies! It’s 2012 and this year Bioline: The PCR Company celebrates its 20 year anniversary.

The world has changed rapidly and dramatically since Bioline was first established in 1992 by Marco Calzavara, president of Bioline, but we are still manufacturing and supplying a range of consistently high-quality products to life science researchers and institutions around the world.

From our base in London, UK, we have rapidly expanded to become a leading primary manufacturer and distributor of PCR enzymes and reagents for molecular biology; providing complete solutions to researchers in universities, major research institutions, hospitals, biotech firms, diagnostics and pharmaceutical firms and laboratories globally. Bioline’s products have been cited thousands of times in leading journals by researchers all over the world.

When asked about the milestone of two decades of service provision to life science, Marco Calzavara, founder and president of Bioline, commented:

“Our aim at Bioline for the past 20 years has been to develop high-quality and reliable products that really make the difference, delivering consistent results time after time. I am excited and proud to continue Bioline’s great work as a cutting-edge molecular biology company, ensuring that we maintain our outstanding reputation as a high-quality and reliable firm.”

Richard L. Eberly, chief commercial officer of Meridian Bioscience, Inc., added:

“Congratulations to the Bioline team on its 20th anniversary of successful operations throughout the world. We are enthusiastic about the success of the merger and impressed by the scientific capabilities of Bioline, as well as its well-deserved reputation for innovation and quality. We remain committed to expanding the rapidly growing portfolio of highly-specialized molecular biology products from Bioline that enable the development of genomic tests utilized by researchers, clinical diagnostic laboratories, diagnostic test manufacturers and biotechnology companies.”

As part of the 20 year celebrations, we are running a monthly Mystery Prize Giveaway for the rest of 2012. Lucky customers will find a golden ticket hiding in their delivery, so keep a close eye out when you receive your delivery. You may find one included with your next order!

We’d like all our customers over the years to join in the celebration. You can do so by visiting the special Bioline 20 Year Anniversary page, where we have a timeline listing the major milestones over the last 20 years and an exclusive interview with our president, as well as other Bioline employees. You can also keep up to date with all the latest 20 year anniversary news and updates by following @BiolineUSA on Twitter and by joining us on Facebook.

Thank you to all of our customers, both old and new alike. We are looking forward to supplying you with more high-quality enzymes and reagents for the next 20 years!

How to Take Your PCR From 11 Hours to TWO

Fides Lay

We caught Fides Lay in the lab on the day of a basketball game between UCLA and crosstown arch-rival USC. As a former UCLA undergrad now working on her PhD at USC, she never wants to miss a game between the two. Unfortunately, the long hours in the lab don’t always allow her to be home in time to watch. At least, that’s how it used to be for her. Thanks to Bioline’s MyTaq HS Mix, now she doesn’t miss a game.

In Peter Jones’ lab at USC, Fides is part of a team studying the epigenetic regulation of cancer. And as anyone who ever worked on epigenetics knows, you need to be a master of PCR to get any results at all. When you need to amplify bisulfite converted DNA to measure methylation status, you work with small samples that often have been digested with several enzymes, with DNA that is fragmented and damaged.

For the longest time the lab amplified bisulfite converted DNA with a tedious protocol. Tedious because of the long set-up with GoTaq®, DMSO and other components added one at a time, and extremely slow cycling with a PCR protocol that was 7-11 hours long. Anything could go wrong at any time, and the result was impossible to predict. Simply using a different thermocycler with a different ramping speed could cause the reaction to fail. And once you found out, it would be too late to fix because every new attempt takes a whole other day. Since the lab usually clones and sequences the PCR fragments, there was always still the risk that no clones would have inserts. Hard to predict, and that meant starting over.

All that changed when Fides first tried Bioline’s MyTaq HS Mix, an easy, all-in-one mix that contains the enzyme, dNTPs, buffer and all optimizers. There’s no need to add any DMSO, it works right away on almost all templates. It’s fast. Reactions are done in less than two hours even on bisulfite converted DNA, with very consistent results and always with nice bands. The PCR products are much easier to clone, and on the rare occasion that something does go wrong, there’s still time to redo the experiment AND get the samples off to pyrosequencing the same day.

Now Fides has time to run multiple experiments and redo anything that goes wrong, all in time to get home, kick up her feet and watch the game.

Zebra Chips with a Side of SensiFAST

In early 2000, a new disease affecting potatoes was discovered in the lower Rio Grande valley areas of Texas. By 2004-2005 the disease spread to other states in the U.S. and was causing significant economic damage in these areas.

The disease is characterized by yellowing of leaves, aerial tubers, shortened and thickened internodes, stunted plant growth and reduced fruit and tuber size. Furthermore, fresh tubers showed brown discoloration when cut and when fried for potato chips they displayed very dark stripes. Hence, the disease was called “zebra chips,” for the characteristic brown stripes. In 2008, an insect, potato/tomato psyllid, Bactericera cockerelli was found to transmit the bacterial pathogen, “Candidatus (Ca.) Liberibacter solanacearum” the casual agent of zebra chip disease in the potato.

Dr. Punya Nachappa is a post-doctoral research associate in Dr. Cecilia Tamborindeguy’s laboratory in the Department of Entomology at Texas A & M University. One of her research projects involves investigating the effects of the bacteria on the insect vector, potato psyllid. Her research shows that the bacteria reduce the reproduction or population of the insects. By using the Bioline SensiFAST SYBR Hi-ROX kit she is able to determine the amount of bacteria in each psyllid and was then able to correlate the amount of bacteria with degree of reproductive loss in the insects. SensiFAST proved to be a reliable product for use in this study.

Research Review: The MyTaq™ DNA Polymerase Family

Below find a compilation of peer-reviewed publications citing use of the MyTaq™ DNA Polymerase family.

MyTaq™ DNA Polymerase
Interesting set of papers for detecting the spread of novel genes from veterinary viruses.

Cadar, D., et al. Virus Genes doi: 10.1007/s11262-011-0650-4 (2011) – Genetic detection and analysis of porcine bocavirus type 1 (PoBoV1) in European wild boar (Sus scrofa)

Cadar, D., et al. Arch. Virol. doi: 10.1007/s00705-011-1125-6 (2011) – Distribution and genetic diversity of porcine hokovirus in wild boars

MyTaq™ Mix
Study of woodland population genetics by 454 shotgun pyrosequencing & PCR. Lead author of the paper, Dr Olivier Lepais, commented:

“We used MyTaq to screen for suitable microsatellite markers in simplex PCR. We found that MyTaq Mix gave great results and was very easy to work with. This ease of use was critical as the primer screening involved a high number of independent PCR mixes!”

Lepais, O. L. & Bacles, C. F. J. Hered. doi: 10.1093/jhered/esr062 (2011) – De Novo Discovery and Multiplexed Amplification of Microsatellite Markers for Black Alder (Alnus glutinosa) and Related Species Using SSR-Enriched Shotgun Pyrosequencing

MyTaq™ Red Mix
Study of microsatellite markers to study the genetic diversity of lobsters in Australia and New Zealand.

Thomas, L. & Bell, J.J. Conserv. Gene. Resources doi: 10.1007/s12686-011-9537-x (2011) – Characterization of polymorphic microsatellite markers for the red rock lobster, Jasus edwardsii (Hutton 1875)

MyTaq™ HS DNA Polymerase
Study on new molecular genetics assay for investigating harmful algal blooms, Sydney Institute of Marine Sciences.

Murray, S.A., et al. Appl. Envir. Microbiol. 77, 7050-7057(2011) – sxtA-Based Quantitative Molecular Assay To Identify Saxitoxin-Producing Harmful Algal Blooms in Marine Waters

MyTaq™ HS DNA Polymerase
Protocol on preparing ssDNA from PCR products with streptavidin magnetic beads.

Wilson, R. Nucleic Acid Therapeutics doi:10.1089/nat.2011.0322 (2011) – Preparation of Single-Stranded DNA from PCR Products with Streptavidin Magnetic Beads

MyTaq™ HS Mix
Interesting new veterinary parasitology paper from the University of New South Wales, Sydney & Merial, a Sanofi Company.

Šlapeta, J., et al. Vet Parasitol. doi: 10.1016/j.vetpar.2011.03.035 (2011) – The cat flea (Ctenocephalides f. felis) is the dominant flea on domestic dogs and cats in Australian veterinary practices

MyTaq™ HS Mix
Bacteria-triggered histone modification in a fungus showing previously undescribed evidence of Saga/Ada dependent histone acetylation triggered by prokaryotes.

Nützmann, H-W., et al. PNAS 108, 14282-14287 (2011) – Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation

MyTaq™ One Step RT-PCR Kit
Neuroscience paper from Department of Cell & Molecular Physiology, Neuroscience Center & Medical School at University of North Carolina.

Buttermore, E.D., et al. J. Neurosci. 31(22), 8013-8024 (2011) – The Cytoskeletal Adaptor Protein Band 4.1B Is Required for the Maintenance of Paranodal Axoglial Septate Junctions in Myelinated Axons

Thousands of papers cite Bioline products, so if you think we have missed an interesting peer-reviewed publication of yours featuring any of our products, please get in touch by email, Twitter, Facebook, or leave a comment on the blog. You gain valuable exposure and profile for your group or company and we share your discoveries with the life science world.

See How Our Cloning Reagents Compare Against Our Competitors

Have you ever wondered how Bioline’s cloning reagents compare against our competitors’? Click here to view a price per mil comparison against Invitrogen, NEB and Stratagene/Agilent competent cells.

Now that you know how our competent cells compare to the competition, did you also know that all Bioline competent cell orders now include SOC Medium?

Competent Cells

AND now, as an added bonus through May 31, 2012, Bioline is offering FREE T4 DNA Ligase and QuickStick with every competent cell order! Download the Competent Cell Guide at www.bioline.com/cloning

This special offer will be applied automatically to online orders. Please quote discount code COMPLIGASE for phone, email and fax orders. Cannot be combined with any other offer. Limit one Quick-Stick Ligase and one T4 DNA Ligase per order. Offer valid through May 31, 2012.

Order Online: Click Here
Email: orders.us@bioline.com
Toll Free: 888-257-5155
Fax: 508-880-8993

MyTaq DNA Polymerase – Product Overview, Features & Benefits

Bioline’s Senior Global Product Manager, Dr. Steve Hawkins, runs through the features, benefits and specially formulated novel buffer system of MyTaq DNA Polymerase.

About MyTaq

The MyTaq DNA Polymerase product range is a new generation of very high performance PCR products developed by Bioline: The PCR Company, specifically designed to deliver outstanding results on all templates, including complex genomic DNA.

MyTaq is based on the latest technology in PCR enzyme preparation, engineered to increase affinity for DNA, resulting in significant improvements to yield, sensitivity and speed. The enzyme is supplied with an industry-leading novel buffer system, specifically formulated and validated for the unique properties of MyTaq, making it the perfect choice for all of your PCR assays.

For more information please see: http://bioline.com/mytaq