The BiosearchTech Blog

Factors affecting fluorophore performance in qPCR

  

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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 (λ_max) 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 λ_max is set to 1. The calculated fraction is then multiplied by the extinction coefficient at the λ_max 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 λ_max. 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

Stellaris FISH Workshop at the AMP 2011

  

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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. 

The 'Monte Carlo' Effect in qPCR

  

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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 C_T 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

Blogging about Stellaris™ RNA FISH at Singlemoleculefish.com

  

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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:

Biosearch Technologies Announces Release of Custom Stellaris™ FISH Probes

  

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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.

Fluorescence Quenching Mechanisms for Dual-Labeled Probes

  

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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.

Special Summer Offer from Biosearch

  

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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.

Multiplexing with Xanthenes

  

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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.

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.

qPCR Troubleshooting

  

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A "Referral from the Doctor" Blog Article-

Quantitative real-time PCR (qPCR) is based upon the fractional cycle number at which a replicating sample of target DNA accumulates sufficient fluorescence to cross an arbitrary threshold. The threshold is either manually selected or auto-selected to fall several standard deviations above baseline fluorescence and below the plateau phase, where the amplification begins to attenuate. Typically, the threshold is adjusted to the mid-point of the exponential phase of the PCR, at a location suitable for all samples in the experiment. The C_T (cycles to threshold) value for a given reaction is defined as the cycle number at which the fluorescence emission intersects the fixed threshold.

What if your assay is non-exponential?

The first step in qualifying newly-developed primers and probes is to test the assay on carefully quantified controls. Irregularities in the amplification of these controls indicate that some modification to the assay must precede its use upon valuable samples.  The diagrams and statements below represent a few of the more commonly occurring issues and how they may be addressed by considering the information presented. This information is intended to be a starting point for trouble-shooting efforts and is not comprehensive. With regards to protocols, it is always best to follow the recommendations of the manufacturer of the polymerase enzyme. When multiplexing, use a master mix specifically designed for multiplex reactions.

Observation: Exponential amplification in the no template control (NTC)

Potential Causes: Contamination; carried over from reagent manufacture or possibly laboratory exposure to the same target sequence, as in gel electrophoresis

Corrective Steps: Clear work area with 10% Bleach and nuclease-free water; order new reagent stocks; relocate reaction set-up to a clean lab

Observation: Looping of data points during early cycles; high noise at the beginning of recorded data

Potential Causes: Baseline adjustment includes too many cycles; too much starting material

Corrective Steps: Reset Baseline to 3 cycles before the first indication of amplification

Observation: Unusually shaped amplification; irreproducible data; later than expected C_T value

Potential Causes: Poor efficiency during PCR reaction; Difference in primer T_ms is > 5 ^°C producing unequal extension; annealing temperature is too low; unanticipated variants within the target sequence

Corrective Steps: Re-design primers to a different region of the target sequence; keep melting temperatures within 2 ^°C of each other; keep the GC content to between 30%-50%; test assay performance against carefully quantified controls

Observation: Slope of standard curve is more or less than –3.34 and R² value is less than 0.98

Potential Causes: Inaccurate dilutions; standard curve exceeds the linear range of detection

Corrective Steps: Recalculate the standard concentration or gene copy number using a spectrophotometer or other means; make new stock solutions of the control standards; eliminate extreme concentrations and limit range to 5 logarithms; consider using a carrier such as a yeast tRNA in the buffer used to generate the dilution series

Observation: Plateau is much lower than expected

Potential Causes: Limiting reagents; degraded reagents such as the dNTPs or master mix

Corrective Steps: Check calculations for master mix; repeat experiment using fresh stock solutions

Observation: Data values all unexpected

Potential Causes: Samples run out of order; plate inserted backwards; poor primer specificity

Corrective Steps: Re-run samples or plate using extra caution when loading; re-design primers to increase specificity

Observation: Actual C_T is much earlier than anticipated

Potential Causes: Genomic DNA contamination; multiple products; high primer-dimer production; poor primer specificity; transcript naturally has high expression in samples of interest

Corrective Steps: DNAse-treat before reverse transcription; re-design primers to increase specificity; decrease primer concentration; increase annealing temperature; increase ramp rate; test assay performance against carefully quantified controls

Observation: Jagged signal throughout amplification plot

Potential Causes: Mechanical error; buffer-nucleotide instability; poor amplification or weak probe signal

Corrective Steps: Contact equipment technician; warm master mix to room temperature and mix thoroughly before use; allow primers and probes to equilibrate for several minutes at room temperature before use; mix primer/probe/master solution thoroughly during reaction set up; if the amount of probe is low or the signal too weak, the program will magnify the baseline noise; redesign the probe and primer sequences

Observation: Technical replicates are not overlapping and have a difference in C_T values > 0.5 cycles

Potential Causes: Pipetting error; insufficient mixing of solutions; low expression of target transcript resulting in stochastic amplification

Corrective Steps: Calibrate pipettes; use positive-displacement pipettes and filtered tips; mix all solutions thoroughly during preparation and during use; hold pipette vertically when aspirating solutions-sterile technique does not ensure reproducibility when working with small volumes

Observation: Irreproducible comparisons between samples

Potential Causes: Efficiency of amplification is below 88% in one or both samples; differences in efficiency are > 5%; RNA degradation; inaccurate dilutions

Corrective Steps: Re-design primers for one or both genes; repeat experiment with fresh reagents and sample

Observation: No data in selected wells

Potential Causes: Wells not selected for analysis; wrong dye selection for analysis; failed first strand synthesis; no expression of target transcript

Corrective Steps: Check settings for data collection and for data viewing; repeat experiment with new reagents; test assay performance against carefully quantified controls

Observation:   Lower concentrations all overlap

Potential Causes: Limited linear range of detection due to low expression of target transcript; carry-over contamination is obscuring the assay’s limit of sensitivity 

Corrective Steps: Eliminate lowest concentration from the dilution series; create a standard curve that spans a higher concentration range

Observation:  Highest concentrations overlap 

Potential Causes: Limit of detection range

Corrective Steps: Eliminate highest concentrations from the dilution series; create a standard curve that spans a lower concentration range

Observation: Baseline drift

Potential Causes: Degradation of the probe

Corrective Steps: Set software for baseline subtraction; remove DTT from your reverse transcription step

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist

Biosearch Technologies and Intelligent Medical Devices, Inc. (IMDx) Finalize cGMP Oligo Supply and Licensing Agreements for Molecular Diagnostic Testing (IVD) Products

  

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Biosearch Technologies, Inc. (Biosearch), a leading supplier of sophisticated oligonucleotide components to the rapidly growing molecular diagnostics industry, and IMDx (privately held), a developer and manufacturer of innovative, clinically impactful molecular test solutions, today announced that Biosearch has licensed access to the BHQ®, CAL Fluor® and Quasar® patents to IMDx.  In addition, Biosearch will manufacture cGMP oligonucleotides for IMDx for use in human in-vitro diagnostics.

To read the entire story, here's the full press release.