Posted on Mon, Mar 19, 2012 @ 09:54 AM
Biosearch Technologies, Inc. (Biosearch), a leading supplier of sophisticated oligonucleotide components to the rapidly growing molecular diagnostics industry, today announced that the company has launched an improved Stellaris™ Probe Designer. The online, RNA FISH design software provides scientists with a means to design Stellaris RNA FISH probe sets against any mRNA or long noncoding RNA.
To read the entire story, here's the full press release.
Posted on Tue, Mar 06, 2012 @ 01:00 PM
Non-squamous, Non-small cell lung cancer (NSCLC), the most common cause of death in the US and globally, has recently been dealt a significant blow as a result of collaborations among teams from UCSF Thoracic Oncology, Kaiser Permanente Research, and the China Clinical Trials Consortium (CCTC) uniting hospitals and universities across mainland China.
In the United States, 150,000 people die annually from the disease, more than breast, colon and prostate cancers combined; 85% of Americans die of lung cancer within 5 years of diagnosis. Worldwide, nearly 1.4 million people die annually. The major challenge in the treatment of lung cancer is a result of the difficulty in the cancer staging process after surgery. Up until now, staging methods have relied on anatomy: size, location and microscopic appearance of the cancer. Lung cancer staging is based on the tumor-node-metastases (TNM) staging system, adopted for lung cancer in 1973-4 and last updated in 2009. Unfortunately, this staging method does not adequately classify patient risk after initial surgery; 35-45% of Stage I lung cancer patients die within 5 years due to recurrent disease because of the inability to find metastatic cancers at Stage I. Under the current system, a “watch and wait” approach is normally applied to early stage NSCLC patients before additional therapeutic interventions such as chemotherapy, radiation treatments, or additional surgery are attempted in order to circumvent further metastasis.
Map: Lung cancer incidence Worldwide in 2008: Men
from GLOBOCAN 2008 (WHO International Agency for Research on Cancer (IARC)) , Section of Cancer Information (29/2/2012)
Dr. David Jablons, Chief of Thoracic Surgery at UCSF, and Kaiser Permanente’s Research team in Oakland, CA, led by Dr. Stephen Van Den Eeden, conducted a blinded study of lung biopsies from 433 patients in Northern California with Stage I cancer. A similar blinded study was concurrently conducted at China’s CCTC on 1,006 lung cancer patients. Both blind studies used quantitative PCR (qPCR) to analyze the expression of 14 genes in FFPE and paraffin-embedded tumor tissues: 11 genes are cancer-related, 3 are housekeeping genes. The staging algorithm used in the study accurately differentiated patients with disease at Stages I, II and III into 3 groups: high, intermediate or low risk. Application of this test will assist physicians with earlier assessment of the advisability of adjuvant treatment regimens beyond surgery.
The test is being offered commercially by a startup, Pinpoint Genomics, through their CLIA-certified laboratory facility in Mountain View, CA. The company is also developing additional diagnostic products to assist in the prediction of the clinical response of individual tumors to different chemotherapeutic regimens.
Biosearch Technologies is proud to be the supplier of the custom probes and primers used in the qPCR assays that helped power this research effort. We are pleased to see the significant strides being made in the field of oncology and look forward to being a part of the world’s fight against cancer.
For more information:
Learn how Biosearch Technologies can support your multiplex diagnostic test development by visiting: http://www.biosearchtech.com/gmp-and-commercial-services/oligonucleotide-manufacturing/ivd-and--molecular-dx
A practical molecular assay to predict survival in resected non-squamous, non-small-cell lung cancer: development and international validation studies
The Lancet, Early Online Publication, 27 January 2012 doi:10.1016/S0140-6736(11)61941-7
The New Lung Cancer Staging System
CHEST July 2009 vol. 136 no. 1 260-271, doi: 10.1378/chest.08-0978
Revisions to the TNM Staging of Non–Small Cell Lung Cancer: Rationale, Clinicoradiologic Implications, and Persistent Limitations
RadioGraphics January 2011 31, 215-238, doi: 10.1148/rg.311105039
Genes used in the prognostic algorithm:
11 Cancer‐related target genes: BAG1, BRCA1, CDC6, CDK2AP1, ERBB3, FUT3, IL11, LCK,RND3, SH3BGR, WNT3A
3 reference genes; (ESD, TBP, YAP1)
Posted on Wed, Jan 11, 2012 @ 11:00 AM
A "Referral from the Doctor" Blog Article-
In qPCR, fluorescent modified oligos may be either linear or non-linear in structure. In either conformation, Förster resonance energy transfer (FRET) combined with the static quenching mechanism inhibits reporter dye emission until hybridization with the target (Read Quenching Mechanisms in Probes to learn more about FRET and static quenching). Upon hybridization the quencher and reporter dye are separated spatially, interrupting the quenching mechanisms and permitting fluorescence emission from the reporter dye. The increase in signal intensity with repeated PCR cycles indicates the accumulation of product and allows for accurate quantification of template. Gene expression analysis by qPCR is dependent upon direct comparisons between the normalized fluorescence emission of individual samples against a standard curve or through ‘relative’ comparison against expression of an internal calibrator gene.
How do I know how bright the dye will be?
- Absolute Intensity - The absolute intensity of a fluorophore is directly proportional to the product of its extinction coefficient and quantum yield. However, the principal determinants of fluorescence detection are the instrument optics and the reaction conditions.
- Excitation sources – for qPCR include: argon lasers which excite at 488 nm and 545 nm; lamps, (halogen or xenon) that emit between 350 nm and 700 nm but at a lower intensity than lasers; or light-emitting diodes (LED) which provide a narrow bandwidth of light but at various wavelengths depending on the LED.
- Dye excitation – or the amount of light a dye can absorb, is directly dependent upon the excitation source and whether it aligns to the dye absorption spectrum. If the excitation source emits at wavelengths far removed from the maximal absorption wavelength (λmax) of the dye, then the extinction coefficient will be some fraction of its maximum and dye intensity will not reach full potential. Be certain that the excitation source uses a wavelength appropriate to excite the dye you have selected.
- Molar extinction coefficient – is the measure of a dye’s light absorption at a particular wavelength and is significant in determining how much incident light will be converted to fluorescence emission. Many common fluorophores have extinction coefficients at their wavelength of maximal absorption between 5,000 and 200,000 mol-1*cm-1. Extinction coefficients may be determined at any place on the absorption spectrum by calculating what fraction of the maximum absorption is occurring at the new wavelength. This is done using normalized absorption spectrum such that the value at the λmax is set to 1. The calculated fraction is then multiplied by the extinction coefficient at the λmax to give the anticipated extinction coefficient at the new excitation wavelength.
- Excitation and emission filters – are carefully engineered to selectively detect the desired dye to the exclusion of adjacent fluorophores. With most organic fluorophores, the maximal emission wavelength is typically around 20 nm from the λmax. The diagram below is a representation demonstrating that qPCR machines have a limited range through which each filter collects fluorescence. It is essential that the reporter dye be aligned with one filter set of the machine.
- Multiplexing – is the simultaneous assay of multiple gene targets, with each gene-specific probe labeled with a different fluorophore. The deconvolution of independent signals may be complicated by fluorescent bleed-through between adjacent channels, or cross-talk. If unanticipated, crosstalk can produce false positive amplifications and impair quantification. The instrument anticipates a certain level of cross-talk based on the dye calibration settings in the software. To avoid cross-talk, select dyes with emission far removed from one another. It may be necessary to calibrate the machine for the emission of the particular dye to be used in the multiplex assay. Some cross-talk is unavoidable.
- Quantum Yield - The inherent flexibility of the dye structure will play a role in the dye quantum yield. Very flexible dye molecules will have lower quantum yields, particularly at higher temperatures, while very rigid dye molecules will have higher quantum yields. Quantum yield may also be dependent on oligonucleotide length, sequence composition, salt concentration and buffer formulation. These dependencies vary from one dye to the next. With FAM-labeled oligos in particular, intensity decreases with increasing temperature or decreasing pH.
Assay performance is different than dye performance. When considering the variables affecting assay performance and its diagnosis when things go amiss, the failure may not necessarily relate to the dye or even the probe but rather other elements in the ensemble, such as primers, enzymes or inhibitors that were unanticipated.
Written by: Christina Ferrell, Ph.D.
References:
Handbook of Fluorescence Spectroscopy and Imaging_John Wiley & Sons_2010
http://www.biosearchtech.com/multiplexing
Posted on Tue, Nov 15, 2011 @ 03:53 PM
Join us at AMP 2011 on November 16th for a live demonstration of the Stellaris technology at the Gaylord Texan Resort in Grapevine, TX. Biosearch Technologies’ lead Stellaris research scientist, Arturo Orjalo, PhD will demonstrate the preparation and fluorescent imaging of single molecule messenger RNA in fixed cells. The workshop will take place in the Texas 1 room at 1:00PM.
Localize and Quantify mRNA with Stellaris FISH Probes Workshop at AMP 2011 Annual Meeting
Presented by: Arturo Orjalo, PhD
Time: November 16, 2011 at 1-2 PM
Location: Grapevine 2 Room at the Gaylord Texan Convention Center, Level 3 in Grapevine, Texas
Can't make the workshop? Stop by our booth.
We'd be happy to answer any questions about the Stellaris technology, or our long-running qPCR products.Stop by booth #518 and take our survey for a free 1GB USB drives loaded with information on Stellaris probes and their applications.
Posted on Wed, Nov 09, 2011 @ 01:00 PM
A "Referral from the Doctor" Blog Article-
The mathematical model of PCR is based on the assumption that each DNA template molecule is reliably duplicated once per cycle, assuming an excess of reagents. In practice, a variety of factors impact amplification kinetics, particularly when the copy number is low, causing deviation from the ideal model. These factors include RNA quality, residual inhibitors, operator technique, primer quality, and efficiency of reverse transcription, to name a few. In addition to those readily identified factors there is also the unavoidable 'Monte Carlo' effect.
The ‘Monte Carlo’ effect, so named because of its association with probabilities to predict outcomes in gambling, describes an inherent limitation while amplifying templates expressed at very low levels. The higher variance in the results from PCR reactions with a low starting template number (< 100 copies) contributes to this statistical phenomenon, such that more qualitative information is produced, as compared to reactions with abundant targets.
An operating theory for this abrupt increase in variation is based on the premise that a primer annealing and successfully replicating an individual template is random and governed by a probability of occurring. If a primer fails to bind then that molecule will not replicate and must await the next cycle for another opportunity. Such a misfire is easily overlooked when millions of other template molecules were successfully replicated during that same cycle, but the consequences are much more pronounced with only one or a few molecules. For this reason, the Monte Carlo effect is presumed to come into play early during thermal cycling, before the target has become enriched. The outcome can be a reduced yield of PCR product or scattered CT values between replicate qPCR reactions.
Such stochastic events must be properly accounted for while interpreting PCR results from samples with low template number or risk compromising quantification. Many more replicate reactions are required to achieve statistical confidence in the results.
Written by: Christina Ferrell, Ph.D.
References:
- Biophysical Journal 71:101-108, 1996
- J Biomol Tech. 15(3): 155–166, 2004
- Proc. Natl. Acad. Sci. USA (Biochemistry) 92:3814-3818, 1995
Posted on Mon, Oct 24, 2011 @ 02:24 PM
Biosearch Technologies is starting a new sister blog at singlemoleculefish.com where all articles will focus on topics related to Stellaris RNA FISH. We will be doing our best to provide as many resources as possible to help you understand the potential of this new RNA FISH technology. You can keep up with the latest by attending AMP's 2011 Annual Meeting at Grapevine, TX. At this molecular pathology conference, Biosearch will be conducting a Stellaris FISH workshop on Weds, November 16 at 1PM in room Grapevine 2.
Check out the introductory blog article and see which other conferences you can catch Biosearch and learn more about Stellaris FISH probes.
Still need to Stellaris RNA FISH? Here's a quick overview to the Stellaris RNA FISH method:
Posted on Mon, Sep 12, 2011 @ 06:59 AM
Stellaris FISH advances research in cancer, developmental biology and pathology by resolving in situ messenger RNA into clear focus.
Biosearch Technologies, Inc. (Biosearch), a leading supplier of sophisticated oligonucleotide components to the rapidly growing molecular diagnostics industry, today announced the release of an empowering new product, Stellaris FISH Probes*. Stellaris FISH (fluorescence in situ hybridization) is a RNA visualization method that allows simultaneous detection, localization, and quantification of individual mRNA molecules at the sub-cellular level in fixed samples.
To read the entire story, here's the full press release.
Posted on Wed, Jun 29, 2011 @ 09:44 AM
A "Referral from the Doctor" Blog Article-
Fluorescent probes are used in biochemical assays to monitor specific events such as binding, cleavage or conformational changes. Dual-labeled probes with a fluorophore and a quencher have many applications in genetic analysis. The efficiency of fluorescence quenching is very distance dependent – if the reporter fluorophore and quencher are far apart, there is fluorescence; if the reporter and quencher are close together in space, fluorescence is suppressed. Typically, the reporter and quencher are placed at specific sites in an oligonucleotide such that a change in their distance will produce a maximal change in fluorescence and effectively signal the event being monitored (often hybridization or nuclease activity). The oligonucleotide acts as a flexible tether linking the fluorescent reporter and quencher. Below, we present fluorescence quenching mechanisms for dual-labeled oligonucleotides in genetic analysis.
I. Static Quenching (also known as contact quenching):
Static quenching involves physical interaction between a reporter and quencher dye resulting in a new, non-fluorescent species called an intramolecular dimer. The resulting dimer is a ground-state complex which has its own distinct features, such as its absorption spectrum. Static quenching efficiency depends on the affinity of the reporter and quencher for each other.
II. FRET (Förster Resonance Energy Transfer) Quenching:
FRET is a through-space mechanism in which energy from the reporter dye is transferred to the quencher without absorption or emission of light.
Efficient FRET requires:
a) Proximity: the donor and acceptor molecules must be close to each other (approx. 10 – 100 Å). FRET is extremely distance dependent.
b) Spectral overlap: the absorption spectrum of the acceptor must overlap with the emission spectrum of the donor.
c) Relative donor-quencher orientation: in most assays with fluorescent probes, it is assumed that the relative orientation of the dyes is random.
Key equation for FRET: 
Where E is the efficiency of FRET energy transfer, R0 is the Förster distance which is the donor-acceptor distance at which energy transfer is 50%, and r is the distance between the donor and acceptor.
These mechanisms may operate independent or simultaneously. For more information on fluorescence quenching mechanisms, please visit our website at: http://www.biosearchtech.com/support/applications/quenching-mechanisms-in-probes.aspx
Written by: Mary K. Johansson, Ph.D., and Christina Ferrell, Ph.D.
References:
Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Salvatore A. E. Marras, Fred Russell Kramer and Sanjay Tyagi. Nucleic Acids Research, 2002, 30, e122.
Intramolecular Dimers: A New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes. Mary Katherine Johansson, Henk Fidder, Daren Dick and Ronald M. Cook. J. Am. Chem. Soc. 2002, 124, 6950.
Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers. Johansson, M.K. Methods in Molecular Biology, v. 335; V.V. Didenko, Ed; Humana Press: Totowa, NJ, 2006; pp 17-29.
Intramolecular Dimers: A New Design Strategy for Fluorescence-Quenched Probes. Johansson, M.K.; Cook, R.M. Chem.-Eur. J. 2003, 9, 3466.
Posted on Mon, Jun 13, 2011 @ 10:49 AM
Despite the unusually brisk May/June weather in California, Biosearch is welcoming the summer with a new promotion to heat things up. Customers now have a chance to receive a $25 Amazon Gift Card and a chance to win an Apple iPad 2 by placing an online order of $750 or more at Biosearchtech.com. For each additional $750+ online order, customers can receive another $25 Amazon Gift Card and another entry in our iPad 2 raffle. Visit Biosearch’s promotions webpage to learn more details.
We hope this promotion will encourage more customers to order through our website, which is regularly updated with new features and improvements. Biosearchtech.com is robust and simple, especially if using an online account. You can order any of Biosearch’s research products online, experience faster checkouts, access multiple saved carts, and track your online order history. Click here to create a free online account at Biosearchtech.com if you’d like to check out these features.
Posted on Wed, Jun 08, 2011 @ 11:13 AM
A "Referral from the Doctor" Blog Article-
Quantitative real-time PCR (qPCR) was developed as a means to detect the presence of target nucleic acids and is routinely used to quantify gene expression through reverse transcription of RNA transcripts. The detection method depends on a signaling molecule, often a dual-labeled oligonucleotide with a fluorophore (reporter) dye at the 5’ end and quencher dye at the 3’ end. The development of new reporter dyes, true dark quenchers and advancements in probe sophistication enable researchers to improve their genetic analysis. One significant improvement is in the application of multiplexed qPCR to yield additional data and confidence in the results.
Multiplexed qPCR combines several PCR assays together into one, to conserve sample material and avoid well-to-well variation. The targets are amplified simultaneously but detected independently using reporters with distinct spectra. Fluorescent dyes used to label oligos are commonly based on the xanthene class of chromophores, which include fluorescein and rhodamine derivatives such as FAM, TAMRA, HEX, JOE, ROX and Texas Red® dyes. Below are the chemical structures for xanthene and its popular market derivative, fluorescein.
Biosearch pioneered the development of new dyes and quenchers intended specifically for multiplexing. The CAL Fluor® series of dyes are also xanthene-based chromophores, but purpose-built for oligo labeling. They are formulated into reactive precursors (phosphoramidites and synthesis supports) intended for automated incorporation during synthesis rather than post-synthesis conjugation through an ester coupling. The table below shows Biosearch dyes and comparable dyes on the market.
| Biosearch Dye |
Comparative Dye |
| CAL Fluor Gold 540 |
TET |
| CAL Fluor Orange 560 |
VIC, HEX, JOE |
| CAL Fluor Red 590 |
TAMRA |
| CAL Fluor Red 610 |
Texas Red |
| CAL Fluor Red 635 |
LightCycler® Red 640 |
These CAL Fluor dyes are efficiently manufactured and remain stable through oligonucleotide synthesis and work up. Attachment chemistry linking the CAL Fluor dyes to biomolecules eliminates the problem of multiple isomers. This results in dye labels that are easier to manufacture, have a single RP-HPLC peak and well-defined emission spectra. Their emission wavelengths span the spectrum to best accommodate the range of optics found in real-time thermal cyclers. When combined with the Black Hole Quencher® (BHQ®) dyes, CAL Fluor dyes offer high signal to noise ratios and reproducible results in multiplex qPCR.
Written by: Christina Ferrell, Ph.D., Technical Applications Specialist
References:
http://www.biosearchtech.com/assets/bti_CAL_Fluor_Dyes.pdf
http://www.biosearchtech.com/assets/bti_bhq_quenching.pdf
http://www.biosearchtech.com/assets/bti_bhq_selectionchart.pdf
For full trademark disclosure, please visit http://www.biosearchtech.com/legal.