FAQs

Real-Time PCR

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• What is “Real-Time” Polymerase Chain Reaction (PCR)?

  Real-Time PCR uses various fluorescent detection chemistries that allow you to monitor the PCR reaction as it progresses. The amount of fluorescent signal generated is directly proportional to the amount of DNA being synthesized during the PCR reaction. Data are collected at each cycle as opposed to traditional PCR, which collects data at the end of the reaction. This allows samples to be characterized by when amplification is first detected as opposed to the amount of product generated after PCR cycling. The greater the amount of the target sequence, the earlier amplification will be detected.

• What are the advantages of Real-Time PCR over traditional PCR?

  One advantage of Real-Time PCR over traditional PCR is that it is a closed-tube system requiring no post-PCR processing. Real-Time PCR has higher precision, increased sensitivity (down to one copy), increased dynamic range (greater than 8 logs), and high resolution (less than two-fold differences).

• What are the applications of Real-Time PCR?

  Real-Time PCR has been used in quantification of gene expression, viral quantification, validation of array data, pathogen detection, and allelic discrimination.

• What are some basic Real-Time PCR terms and their definitions?

  Some basic Real-Time PCR terms and their definitions are:

  Amplification plot—Plot of fluorescent signal versus cycle number.

  Baseline—The initial cycles of PCR where there is little to no change in fluorescence.

  Threshold—The arbitrary level of fluorescence used for Cq determination. Should be set above the baseline and within the exponential growth phase of the amplification plot.

  Cq (quantification cycle)—The fractional cycle number where fluorescence increases above the threshold. Also referred to as Ct (threshold cycle) or Cp (quantification cycle).

  R—Reporter signal.

  Rn—Normalized reporter signal.

  ΔRn—Baseline subtracted normalized reporter signal.

  Slope—Indicates the efficiency of the reaction. With 10-fold dilutions, a slope of -3.32 indicates a perfect doubling of product per cycle (100% PCR efficiency).

  R2—Reports the linearity of the standard curve.

• What are the phases of PCR amplification?

  There are three phases of PCR amplification: exponential, linear, and plateau. The exponential phase is the first phase of PCR amplification. Reaction components are in excess, there is an exact doubling of product each cycle, and the reaction is specific and precise. Real-Time PCR measures the Cq value at this phase of PCR. The linear phase is the second phase of PCR amplification. The reaction components are being consumed, amplification slows, and the reactions become highly variable. The final phase of PCR amplification is the plateau phase. The reaction is complete and no more products are being generated. Traditional PCR takes its measurements during this phase of PCR.

• I am currently running traditional PCR; can I use the same template, primers, and reagents to run Real-Time PCR?

  In some cases it is possible to convert existing traditional PCR assays into Real-Time PCR assays, with a few considerations around primer design and master mix. Primer design is one of the first considerations for converting a traditional PCR assay. Real-Time PCR is most efficient with relatively short amplicon lengths, in the range of 50 to 150 bp. Longer products can be used if the cycling conditions are changed to accommodate longer extension times, but you should avoid products longer than 300 bp. In some cases it might be possible to design a TaqMan probe to hybridize between the two existing PCR primers. If not, you can use SYBR Green I for detection. (See other FAQs for information on TaqMan and SYBR Green I.)

  The master mix is another consideration when converting a traditional PCR assay into a Real-Time PCR assay. If a TaqMan probe can be designed, you might be able to use the same master mix that was used for the traditional PCR assay. If a TaqMan probe cannot be designed, you should add SYBR Green I to the master mix. In either case, a certain amount of optimization may be needed to obtain good Real-Time PCR results.

• Does Illumina sell Real-Time PCR reagents?

  No, Illumina does not currently sell Real-Time PCR reagents.

• What are the major detection chemistries used for Real-Time PCR?

  There are two major detection chemistries used for Real-Time PCR: hydrolysis (TaqMan) probe-based chemistry and DNA-binding SYBR Green I dye-based chemistry. Less used detection chemistries include Molecular Beacons, Scorpion probes and LUX primers.

  In TaqMan probe-based chemistry, also known as the fluorogenic 5’ nuclease assay, an oligonucleotide probe anneals to a specific sequence downstream of one of the PCR primers. The oligonucleotide is labeled with a fluorescent reporter dye at the 5’ end and a quencher dye at the 3’. When the probe is intact, the reporter is in close proximity to the quencher and the fluorescent signal is low as the energy from the reporter will be transferred to the quencher through Fluorescent Resonant Energy Transfer (FRET). During PCR, as Taq DNA polymerase extends from the primers, the 5’ exonuclease activity of the enzyme cleaves the annealed probe to separate the reporter dye from the quencher dye, increasing the fluorescent signal.

  SYBR Green I is a dye that binds only to double-stranded DNA (dsDNA) and its fluorescent signal increases only when bound to dsDNA. During PCR, the fluorescent signal of SYBR Green I increases along with the dsDNA amplicon.

• What are the advantages and disadvantages of TaqMan and SYBR Green I chemistries?

  The main advantage of TaqMan chemistry is that a fluorescent signal is generated only when there is specific hybridization of the probe to the target sequence. No signal is generated from any non-specific amplification products that were formed during the reaction. Another advantage is that probes can be labeled with different, spectrally distinct reporter dyes, which allows the amplification of multiple target sequences within a single tube (multiplex Real-Time PCR). The main disadvantage of TaqMan chemistry is that design and synthesis of different dual-labeled probes is required for each target sequence, which increases assay setup and cost.

  The main advantage of SYBR Green I chemistry is that it only requires the design and synthesis of two PCR primers, which decreases assay setup and cost. Another advantage for SYBR Green I chemistry is the ability to perform melt curves. The main disadvantage of SYBR Green I chemistry is that since SYBR Green I binds to any dsDNA present during the reaction it will bind to and generate a signal for any non-specific amplification that occurs.

• What is melt curve analysis?

  Melt curve analysis is a post-PCR analysis that is compatible with SYBR Green I-based assays. During a melt curve, amplicons produced during PCR are dissociated (or melted) by slowly ramping the instrument from a low temperature to high temperature. The fluorescence is monitored throughout. As the amplification products transition from dsDNA to single-stranded DNA and the SYBR Green I is no longer bound, there is a sharp decrease in fluorescence. The midpoint of this transition is known as the melting temperature (Tm) of the sample and is characteristic of a given DNA sequence. Melt curves are useful for determining the specificity of a PCR reaction, as any non-specific amplification products will have a different melt curve profile than the target sequence.

• 1) Are SYBR Green I Real-Time PCR assays less specific than TaqMan probe assays?

  The specificity of any Real-Time PCR assay, whether TaqMan probe or SYBR Green I, is determined by the quality of the assay design. Non-specific amplification can occur for both SYBR Green I or TaqMan probe methods if the assay design is poor.

  TaqMan assays will not generate a signal for any non-specific amplification whereas SYBR Green I assays might. However, non-specific amplification will affect the amplification efficiency and sensitivity of TaqMan assays in the same way as SYBR Green I assays, even though the amplification is not detected.

  When designing primers for either system, it is important to avoid primer sets that generate any non-specific amplification products. With SYBR Green I assays, the ability to perform melt curve analysis is advantageous for primer design, as any non-specific amplification can be detected and identified in the melt curve. For TaqMan assays, detecting non-specific amplification usually requires another post-PCR analysis method such as agarose gel electrophoresis of the PCR products. Alternatively, the primers could be used in PCR with SYBR Green I and melt curve analysis performed after amplification to determine if any non-specific amplification occurs. This optimizes the primer design without the expense of a labeled probe.

• How do I design primers/probes for a Real-Time PCR assay?

  There are numerous primer design tools commercially available for purchase or freely accessible via the web. These tools simplify assay design significantly. Some widely used primer design tools are Primer Express (Applied Biosystems), Beacon Designer (Premier Biosoft) and Real Time Design(BioSearch Technologies). Numerous websites contain databases of validated primer sets, including RTPrimerDB (http://medgen.ugent.be/rtprimerdb/) and the Quantitative PCR Primer Database (http://web.ncifcrf.gov/rtp/gel/primerdb/)

  If you design primers and probes manually, follow these criteria from the Primer Express manual:

  • The amplicon length should be between 50–150 bp to maximize amplification efficiency.
  • The optimal primer length is 20 nucleotides and the Tm should be between 58° and 60° C.
  • The optimal probe length is between 13 and 30 nucleotides for standard TaqMan probes and the Tm should be between 68° and 70°C.
  • Primer and probe sequences should not overlap.
  • The GC content of primers and probes should be between 30% and 80%.
  • To prevent quenching, the 5’ end of the probe cannot be a G residue.
  • Avoid runs of identical nucleotides. Primer and probe sequences must contain fewer than four consecutive G residues.
  • Ideally, to avoid genomic DNA amplification, primers should span an exon-exon junction in cDNA.

  Additionally, you should perform a BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) on primer and probe sequences to determine if the primers/probe will amplify sequences other than the target sequence.

• What are the different starting materials that I can use for Real-Time PCR?

  The starting materials for Real-Time PCR can be RNA, genomic DNA, or plasmid DNA. RNA must be reverse-transcribed into complementary DNA (cDNA) before PCR. More recently, kits have become available that can perform RT-qPCR (RT = Reverse Transcription) directly from cells without the need for a separate RNA synthesis step.

• What dyes and specific wavelengths does Eco support?

  The following table details the excitation and emission wavelengths of Eco in each channel. The emission filters combined with spectral de-convolution algorithms effectively minimize cross-talk between dyes. Eco is factory calibrated for SYBR Green I, FAM, HEX, VIC, ROX, and Cy5. You can use other dyes if they are within the wavelength range of the emission filters.

  Channel

  Excitation (nm)	Emission (nm)	Example Fluorophores Detected
  1	452–486	505–545	SYBR Green I, FAM
  2	542–582	604–644	ROX
  3	452–486	562–596	HEX, VIC
  4	542–582	665–705	Cy5

• The Eco system supports standard dyes used for multiplexing; can I use other dyes or new dyes in the future?

  The Eco optical system supports dyes within four channels, ranging from 505 to 705 nm. The optical system can detect any dyes that fall within those ranges.

  Channel

  Excitation (nm)	Emission (nm)	Example Fluorophores Detected
  1	452–486	505–545	SYBR Green I, FAM
  2	542–582	604–644	ROX
  3	452–486	562–596	HEX, VIC
  4	542–582	665–705	Cy5

Multiplex Real-Time PCR

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• What is multiplex Real-Time PCR?

  Multiplex Real-Time PCR is a technique in which multiple target sequences are amplified and detected in a single PCR reaction. Amplified sequences are distinguished by the use of different dyes conjugated to the TaqMan probes. The number of targets that can be detected in a single reaction is technically limited only by the availability of spectrally distinct dyes and the ability of the Real-Time PCR instrument to effectively excite and detect those dyes. Some advantages of multiplex Real-Time PCR are reduced reagent costs, reduction in sample use, and increased throughput.

• What is required in developing multiplex Real-Time PCR assays?

  Developing multiplex Real-Time PCR assays can be difficult and time-consuming. As the reaction complexity increases, significant optimization may be required to generate reliable data. It can be a challenge to develop multiplex assays that amplify all targets with equal efficiency.

  When developing multiplex Real-Time PCR assays, you need to consider primer design, the relative expression levels of target sequences, and master mix / reagent conditions.

  Use the same design criteria for each primer/probe set and screen all sequences against each other to determine any potential primer-dimer formation. In addition, perform a BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine primer specificity.

  If the expression levels of the target sequences are significantly different, the most abundant target will be preferentially amplified and deplete all the reaction components, compromising amplification of the less abundant targets. One way to address this issue is to limit the primer concentrations of the most abundant target, using the lowest primer concentration that produces the same Cq and PCR efficiency. Limiting the primer allows the most abundant target to amplify and go to completion without depleting all the reagents needed for the other sequences.

  Amplifying multiple target sequences creates additional demand for reaction components. Taq DNA polymerase, Mg++ and dNTP concentrations may need to be optimized to improve amplification of all targets. Master mixes optimized specifically for multiplex Real-Time PCR are now commercially available, and can reduce the amount of time required for optimization.

• What are the applications of multiplex Real-Time PCR?

  Multiplex Real-Time PCR can be applied to relative quantification experiments where the gene of interest and reference gene are co-amplified in the same reaction. Multiplex Real-Time PCR can also be used for allelic discrimination assays, where two differentially labeled probes detect two alleles of a single nucleotide polymorphism. Another application of multiplex Real-Time PCR is pathogen detection, where multiple pathogens can be detected in one reaction.

RNA in Real-Time PCR

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• How do I quantify my RNA sample?

  The most widely used method to quantify RNA is traditional UV spectroscopy. A diluted RNA sample is quantified by measuring its absorbance at 260 nm and 280 nm. The concentration is calculated using the equation:

                                                  [RNA] μg/ml = A260 x dilution factor x 40

                                                  where 40 is the average extinction coefficient for RNA

  In addition, the A260/A280 ratio can be used to estimate RNA purity. An A260/A280 ratio between 1.8 and 2.1 indicates a highly pure RNA sample.

  UV spectroscopy is relatively simple to perform but has several drawbacks. It does not discriminate between RNA and DNA so it is advisable to DNAse treat RNA samples before quantifying. DNA in the sample will lead to an overestimation of RNA concentration. Since proteins and residual phenol from the purification can interfere with absorbance readings, it is important to remove these contaminants in purification. Also, absorbance readings are dependent on pH and ionic strength. Dilute RNA samples in TE (pH 8.0) and use TE to blank the spectrophotometer before taking absorbance readings.

  An alternative method for quantifying RNA samples is to use fluorescent dyes such as RiboGreen (Invitrogen). RiboGreen exhibits a strong fluorescent signal when bound to nucleic acids. Samples are quantified in a fluorescence microplate reader or standard spectrophotometer relative to a nucleic acid standard curve of known concentration. The linear range of quantification using RiboGreen is three orders of magnitude, from 1 μg/ml down to 1 ng/ml. The major advantage of fluorescent dyes over absorbence-based methods is that it is not affected by contaminating proteins or organic solvents carried over from the purification process. DNAse treatment is still recommended as RiboGreen does not discriminate between RNA and DNA.

• How do I assess the quality of my RNA sample?

  In Real-Time RT-qPCR, genomic DNA can potentially be co-amplified during the PCR reaction, contaminating the sample and leading to erroneous results. To determine if an RNA sample is contaminated with genomic DNA it is important to include a no-reverse transcriptase control during the RT step, and all RT-qPCR experiments should include a no-RT control. If the RNA sample is free of genomic DNA contamination the no-RT controls should not generate any signal after Real-Time PCR.

  To avoid genomic DNA contamination, treat RNA samples with DNAse before reverse transcription. Alternatively, design the PCR primers to anneal to sequences of the transcript that span a large intron. Primers designed in this way can only amplify cDNA.

• What is the difference between one-step and two-step real time RT-qPCR?

  The difference between one-step and two-step real time RT-qPCR lies mainly in the reverse transcription step. In one-step RT-qPCR, a short reverse transcription (5–30 minute) reaction is followed by a PCR reaction in a single tube. In two-step RT-qPCR the reverse transcription reaction takes place in a separate tube. Each method has advantages and disadvantages, depending on the application.

  One-step real time RT-qPCR, which uses gene-specific primers, is useful when analyzing a few genes over a large number of samples. Since both the RT and PCR reactions occur in the same tube, there is less pipetting and sample manipulation, possibly reducing variation and potential contamination. One-step RT-qPCR might not be as sensitive as two-step since its reverse transcription step is much shorter. The reaction conditions needed to support both the RT and PCR reactions might not be optimal for either reaction. Another drawback of one-step RT-qPCR is that it is not possible to archive the cDNA produced during the reverse transcription reaction.

  Two-step real time RT-qPCR is useful when analyzing a large number of genes over a few samples. It supports a flexible priming strategy, allowing for oligo-dT, random primers, or gene-specific primers. Two-step RT-qPCR is generally more sensitive than one-step since the RT reaction is much longer and the RT and PCR reactions occur separately, meaning that they can be optimized individually. Also, the cDNA produced is more stable than the initial RNA sample and can be more easily archived for future use.

Quantification Assays in Real-Time PCR

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• What is the difference between a relative quantification assay and an absolute quantification assay?

  A relative quantification (also known as comparative quantification) assay quantifies changes in gene expression relative to a reference gene and reference sample. An absolute quantification assay uses a standard curve of known quantities to determine the quantity of unknown samples. Relative quantification results report fold changes in expression relative to the reference gene and reference sample. Absolute quantification results are reported as an absolute quantity (copies, ng, pg, etc.) extrapolated from the standard curve.

• What mathematical model does the Illumina Eco analysis software use for relative quantification experiments?

  The Illumina Eco analysis software uses the ΔΔCq method (Livak et al., 2001), which is the most commonly used analysis method for relative quantification. The ΔΔCq method reports fold change in gene expression of a target gene relative to that of a reference gene and reference (calibrator) sample. The reference gene is typically a housekeeping gene such as GAPDH, β-actin, β2-microglobin or HPRT. The choice of reference sample can vary depending on the type of gene expression experiment. For example, a reference sample might be an untreated control sample relative to a treated sample, normal tissue relative to diseased tissue, or liver relative to brain.

  For the ΔΔCq method to be valid certain assumptions must be met. First, the reference gene must be stably expressed between different cells of tissues and unaffected by any experimental treatment. Second, the amplification efficiencies of both target gene and reference gene must be approximately equal. Both of the conditions should be experimentally validated for each experiment before the ΔΔCq method can be used.

  When extreme accuracy is needed, the Eco software supports use of multiple reference genes as well as PCR efficiency correction.

• What do I need to do if I want to use another method for relative quantification analysis?

  The Eco analysis software does not provide alternative methods of relative quantification analysis, but third-party tools are available. You can export all data in *.csv format from the Illumina Eco analysis software and import the data into third-party analysis software such as REST or qBase.

• How do I choose an appropriate reference gene?

  Selection of a reference gene or genes is a critical step for expression analysis using Real-Time PCR. Validation of reference genes for each experimental condition is critical for obtaining accurate Real-Time PCR data. Validation requires determining if expression of the reference gene is stable between cells of different tissues and if any experimental treatment affects expression.

  The following paper contains a good summary of the validation process:

  Dheda K., et al. Validation of housekeeping genes for normalizing RNA expression in Real-Time PCR. Biotechniques 2004; 37: 112–119.

  The process starts with extracting and quantifyingRNA samples from the samples under investigation (diseased v. normal; treated v. untreated). The next step is normalizing the input of RNA into the reverse transcription reaction. The expression of a panel of different reference genes is then measured by Real-Time PCR and the differences in Cq across the different samples is determined for each gene.

  There are a number of software programs available for selecting appropriate reference genes, including GeNorm (http://medgen.ugent.be/~jvdesomp/genorm/), BestKeeper (http://www.gene-quantification.de/BestKeeper-1.zip) and Norm-Finder (http://www.mdl.dk/Files/NormFinder_0953.xla).

• Is it possible to perform QF-PCR on Eco?

  The initial PCR amplification for QF-PCR (Quantitative Fluorescence PCR) can be performed on Eco. The subsequent detection and quantification of the amplified fragments requires electrophoresis on a DNA scanner. Some applications of QF-PCR, such as copy number variation (CNV) analysis, can be adapted into Real-Time PCR and be performed on Eco.

• Can Eco be used to quantify proteins labeled with fluorescent dyes?

  Eco does not support this application.

High-Resolution Melt (HRM)

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• What is High-Resolution Melt (HRM) curve analysis?

  HRM is a recent advancement to the traditional melt curve analysis that significantly increases the amount of detail and information that can be captured. It is sensitive enough to be able to differentiate sequence differences within PCR amplicons down to a single nucleotide. Mutations within amplicons are detected as either a shift in the Tm of the product or a change in the shape of the melting curve.

• What is required to perform HRM?

  High-resolution melt curve analysis requires a different class of dsDNA binding dyes, extremely precise instrumentation, and specialized software.

  HRM analysis is generally performed using dsDNA binding dyes other than SYBR Green I. These dyes are generally known as saturating dsDNA-binding dyes, and include SYTO 9 (Invitrogen), LCGreen (Idaho Tech), and EvaGreen (Biotium Inc.). These dyes differ from SYBR Green I in that they are significantly less inhibitory to PCR. This reduced inhibition allows them to be used at much higher concentrations than SYBR Green I that saturate the dsDNA amplicons. Greater dye saturation provides greater sensitivity and resolution of melt curve profiles.

  Extremely precise instrumentation is important for HRM. Since some mutations only cause Tm shifts of a fraction of a degree, any thermal or optical non-uniformity will reduce the ability to detect these sequence changes. To be able to perform HRM analysis an instrument needs to have a fast acquisition rate, precise temperature control, and an absolute minimum of thermal and optical variation between samples.

  HRM also requires is software with specialized analysis algorithms that can analyze the shape of melt profiles and group similar melt profiles together. HRM data can be viewed as either normalized melt curves or difference plots. Difference plots show the difference in fluorescence from a selected reference sample. Some software also features an auto-call feature, which can automatically assign genotypes based on melt profiles.

• What are the applications of HRM?

  High-resolution melt curve analysis was originally developed for SNP genotyping but has since been applied to different applications of mutational analysis. Applications for HRM include mutation discovery, DNA fingerprinting, species identification, HLA compatibility typing, allelic prevalence, and DNA methylation analysis.

• How does the Eco system achieve higher HRM performance than other block-based systems?

  Temperature control and uniformity are the most challenging factors affecting HRM. The Tm shift for a single base change can be as small as 0.2° C for challenging Class IV A to T single nucleotide polymorphisms. Most current block-based instruments report temperature uniformity specifications in the range of ± 0.25° C to ± 0.5° C, generally considered too high to reliably differentiate a Class IV SNP. Block-based instruments that claim to perform HRM do so by extensive calibration and software compensation or by employing specialized analysis methods, such as temperature shifting, to overcome the thermal non-uniformity across the block.

  Eco’s unique thermal block provides thermal uniformity of ± 0.1° C, well above the industry standard for a block-based system. This extreme thermal uniformity allows Eco to perform HRM without software corrections. The thermal uniformity of Eco supports genotyping of even the most challenging Class IV SNPs.

The Eco System

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• What chemistries can I run on the Eco Real-Time PCR system?

  Eco is an open platform that supports all current Real-Time PCR detection chemistries including DNA binding dyes, hydrolysis probes, as well as other detection chemistries such as molecular beacons and Scorpion primers.

• Does the Eco Real-Time PCR system require separate software for analysis?

  No, the Eco software provides instrument control, data collection, and data analysis capabilities in a single package. If more advanced analysis is required, you can export all raw data in Excel or CSV format for convenient analysis in other third-party software.

• Why does the Eco Real-Time PCR System come in a 48-well format?

  Our market research indicated that >70% of Real-Time PCR users run fewer than 50 wells per plate. In addition, the small well volume is optimized for a reaction volume between 5–20 µl, while 96-well plates are optimized for 25–50 µl reaction volumes. The 48-well plate enables you to save on the cost of reagents.

• Is the Eco Real-Time PCR system available in block formats other than the 48-well block?

  Currently, Eco is only available in a 48-well format.

• Can I purchase Eco plates from another vendor than Illumina?

  Currently, Eco plates can only be purchased from Illumina. The pricing is aligned with the price of plates on other systems. On average, the price of consumables (plates and seals) is about 20% less expensive than the list price offered by the competition.

   

  COMPLETE PRICE* (US$)	Eco 48‑well	Abgene 96-well	ABI 96‑well	ABI 48‑well	Rotorgene 72‑well	Stratagene 96‑well	BioRad 96‑well	BioRad 48‑well
  Price per well	7.29	10.43	8.57	9.34	13.90	8.27	8.84	8.13
  Price per 48‑well equivalent	3.50	4.99	4.11	4.48	6.67	3.98	4.22	3.88
  Savings with Eco		30%	15%	22%	48%	12%	17%	10%

  * This information reflects U.S. pricing.

• Is the Eco loading dock sold separately?

  One complete loading dock will be provided with each Eco. To purchase additional docks, go to http://www.illumina.com/eco and click the Options and Accessories tab.

• What warranty is provided with the Eco?

  The Eco comes with a one-year warranty that provides full coverage for all service parts and labor, hardware and software updates, and access to online training modules. The warranty includes comprehensive email and telephone support for the instrument, its applications, and its bioinformatics. Service is conducted at the manufacturer site and will be completed within five business days on average.

  Extended warranty and calibration plans are available if needed.

• What is the acquisition time for Eco?

  The acquisition time for all four channels is approximately six seconds.

• What is the temperature resolution of Eco?

  The temperature resolution of Eco is 0.1° C.

• What is the energy consumption of Eco?

  The peak power of the instrument is 500 VA. Typical power consumption during a run is around 180 VA.

• What is the noise level of Eco?

  Eco is extremely quiet, producing less than 62 db during a run.

• What are the Eco’s humidity and temperature requirements?

  Temperature Operating Range         15° to 30° C (59°F to 86° F)

  Storage                                           10° to 38°C (50°F to 100°F)

  Humidity Operating Range               15–90% RH

  Storage                                            5–95%RH

• Does the fluorescent acquisition occur through the side or top of the plate?

  The acquisition is done by a CCD camera through the top of the plate and through the optical adhesive sealer.

• How does the Eco system provide a significantly higher level of thermal uniformity compared to other block-based systems?

  Eco’s hollow thermal block is constructed of highly conductive silver and is filled with a thermal conductive fluid. The fluid is rapidly circulated through the block by paired agitators that are powered by high-efficiency electromagnetic motors. The constant circulation of fluid evenly distributes the heat throughout the block to remove the hot or cold spots typical of block-based instruments. This design allows for rapid temperature ramps and extreme thermal uniformity (± 0.1° C) well to well across the plate, with no edge effect.

• What is the liquid inside the Eco thermal box? Is it dangerous or toxic?

  The fluid contained in the heating block is specifically designed for use in heat transfer applications and has been used for the last 30 years as an electronic coolant. The fluid is hermetically sealed in each block, is environmentally benign, and has a low vapor pressure.

• My current system requires periodic optical calibration to ensure performance; how does Eco address this?

  The Eco optical system components do not move during operation, with the exception of the filter slide, which moves while measuring the four emission wavelengths for each sample at each cycle.

  The optical system is calibrated prior to shipment and does not require recalibration. An optional yearly calibration plan is available. If your application requires annual validation, contact Customer Service at 1.800.809.4566 in the U.S. or email orders@illumina.com for pricing and assistance.

• How long do you expect the Eco LED array to last? Can Illumina provide a lifetime warranty for the LED array?

  The LED arrays are designed to outlast the life of the instrument and are covered by the instrument warranty.

• Are the detector photomultiplier (PMTs) or photodiodes resistant to dark current, shock, and vibration?

  Eco does not use PMTs or photodiodes. It uses a CCD camera as the detector and is designed to compensate for dark current.

• What are the Eco power requirements?

  Eco runs from a standard wall socket over a wide voltage range (100–240VAC). It does not require 3-phase power. Eco ships with a European line cord and a U.S. line cord. The local distributor should provide an appropriate line cord for local power outlet configuration.

• How can Illumina offer the Eco Real-Time PCR system for only $13,900?

  Illumina can offer Eco for only $13,900 through a combination of smart, simple design and engineering. Eco was designed using high-quality off-the-shelf components that require little to no customization, significantly reducing the cost of goods for each instrument. In addition, Eco is not sold through a traditional field sales and support team. All sales and support are managed directly through the Eco website which is more convenient for our customers.

  The low cost of manufacturing, selling, and supporting Eco systems allows Illumina to pass the savings on to our customers and deliver the first affordable high-performance Real-Time PCR system.

• Can I have articles, publications, certificates of testing, or details on research studies using Eco that have been done on the instrument?

  As Eco is a new instrument that has not been released to market, there are currently no publications citing the use of Eco. Some data are available on www.ecoqpcr.com. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.

Technical Support

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• How do I contact Technical Support?

  The fastest way to answer most questions is to refer to the Eco Support web page or in these FAQs. If you do not find an answer there, you can reach Technical Support by email at techsupport@illumina.com, by phone at 1.800.809.4566 in the U.S., or on the Illumina website at http://www.illumina.com/support.

• What type of information should I provide when contacting Technical Support?

  For the fastest, most efficient support, supply your name, institution, contact information, and the serial number from the grey label on the back of your instrument. The warranty, instrument, and previous case history are associated with the instrument serial number, so this information will enable Technical Support scientists to obtain the data they need to support you.

Processing the Data

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• On how many computers can I install the Eco system software?

          The Eco comes with five licenses for the system software, so that you are not constrained to perform the data analysis on the included workstation. You can install the Eco software from the USB drive that is provided with each Eco. If you require more licenses, contact Illumina customer service.

• Can I export my Eco run data for use in other software packages?

  You can export your data as multiple *.csv files or as a single Excel file with multiple worksheets. In addition, you can export a report as a PDF or PowerPoint file.

• How do I determine the efficiency of my Real-Time PCR assay?

  The simplest and most commonly used method is the dilution or standard curve method. This method calculates PCR efficiency using the linear regression slope of a dilution series based on either of the following equations:

  • E = 10(-1/slope) -1
  • E = 10(-1/slope)

  The ideal slope is -3.32, which correlates to an amplification efficiency of 100%, meaning exactly one copy per cycle. Slopes in the range of -3.60 to -3.10 are generally considered acceptable for Real-Time PCR. These slope values correlate to amplification efficiencies between 90% (1.9) and 110% (2.1).

• How can I prevent or reduce amplification in my no-template control (NTC) reactions from SYBR Green I assays?

  Amplification in NTC reactions can either be from contamination or non-specific amplification. Performing melt curve analysis can help identify if the signal is from contamination or from non-specific amplification.

  If it is contamination, the melting curve of the NTC reaction will have the same T_m as your target sequence. Good aseptic technique, using aerosol-resistant pipette tips and a Real-Time PCR master mix with dUTP and UDG, can help to reduce any potential contamination.

  If the signal is due to non-specific amplification, the melting curve of the NTC reaction will have a different T_m than the target sequence. The most common type of non-specific amplification is primer-dimer formation, and there are a number of ways to reduce this.

  Optimal primer design is an important first step in preventing primer-dimer formation.

  • Primer pairs should be screened using oligonucleotide analysis tools for any secondary structure, self-annealing, or cross-annealing.
  • A hot-start DNA polymerase will prevent the extension of non-specific primer annealing during the reaction setup.
  • Decreasing the primer and Mg++ concentrations can reduce primer-dimer formation, but can have a negative effect on amplification efficiency.
  • Additives such as glycerol and DMSO have been known to reduce primer-dimer formation.

  If these steps do not help reduce primer-dimer formation, redesigning the primers is necessary to obtain good results.

Software

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• Does the software allow the user to perform a regular check on the performance of the detection system of Eco?

  Use the Eco evaluation plate to test the performance of the instrument whenever needed.

• Does the Eco software have individual profiles for users with password protection, file storage, and data analysis preference?

  The Eco software was designed for individual users, so it does not provide user profiles or password protection. However, you can configure Windows 7 on the provided netbook for multiple users, giving each user an individual login with personalized settings and folders.

• How many runs can I save in the software?

  You are unlikely to exceed the storage capacity of the provided computer. Eco run files are small (~200–500 kb for a 40-cycle run), while the computer hard drive is 220 GB.

• Can the Eco software generate expression fold changes automatically after the assay completes the run, without further command and analysis?

  The Relative Quantification application calculates and displays fold changes automatically if you defined your reference sample as well as your reference assays. The software automatically calculates and displays error bars for each data point on the graph.

• Is it possible to import standard curves from a previous run?

  That feature is not currently supported. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.